Latent human tuberculosis model, diagnostic antigens, and methods of use

ABSTRACT

Provided herein is an in vitro granuloma model and methods of its use. Methods of detecting and/or diagnosing latent tuberculosis in a subject are also provided, as are latency-specific antigens (and antibodies thereto), such as α-crystallin, and methods of identifying and using such molecules. Also provided are immunostimulatory compositions, for instance for use in eliciting an immune response in a subject, such as an immune response to a latent tuberculosis infection. Kits for carrying out the provided methods are also described.

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/260,348, filed Jan. 8, 2001, and U.S. ProvisionalApplication No. 60/311,235, filed Aug. 9, 2001.

STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made by the Centers for Disease Control andPrevention, an agency of the United States Government. Therefore, theU.S. Government has certain rights in this invention.

FIELD OF THE DISCLOSURE

[0003] The present disclosure relates to the field of mycobacteriallatency, and in particular relates to an in vitro granuloma model forthe study of mycobacteria and for the development of tuberculosis drugand vaccine candidates, and to the detection of latent mycobacterialinfection using immunoassays.

BACKGROUND

[0004] Approximately every ten seconds, a person dies of tuberculosissomewhere in the world. Tuberculosis is the world's number one killeramong infectious diseases and the leading cause of death among women ofreproductive age. Although developing countries bear the greatest burdenof disease, the United States is greatly affected by tuberculosis,reporting 16,377 cases in 2000.

[0005] The infectious agent causing almost all cases of tuberculosis isMycobacterium tuberculosis (M. tuberculosis). M. tuberculosis is easilyspread between individuals through the air. A single cough by aninfected individual can generate as many as 3000 infected dropletnuclei, while less than 10 bacilli may initiate a pulmonary infection ina susceptible individual. Because simply inhaling an airborne pathogenmay infect individuals, tuberculosis outbreaks are difficult to containand require isolating the infected individuals in negative air pressurerooms.

[0006] Although it was believed that tuberculosis would eventually beeliminated after the development of antibiotics in the 1950s, in 1999,tuberculosis was labeled as a global health emergency by the WorldHealth Organization. One of the major reasons for the perseverance oftuberculosis is the evolution of multi-drug resistant strains.Multi-drug resistant strains have evolved in part due to infectedpatients' poor compliance with drug therapy, which lasts for a period ofat least six months. One multi-drug resistant strain, strain W, hasevolved resistance to all first-line drugs (isoniazid, rifampin,ethambutol, and pyrazidine), as well as one second-line drug(kanamycin). It is therefore evident that tuberculosis continues to be aserious health threat to individuals worldwide.

[0007] Initial infection with M. tuberculosis only rarely leads toimmediate disease because the infection is typically controlled by thehost's immune system. Among people infected with M. tuberculosis,approximately 5% manifest the disease within a few years afterinfection. Upon initial infection, the mycobacteria enter unactivatedmacrophages and multiply therein. Following a rapid growth phase,infected macrophages and their bacilliary cargo are surrounded andwalled off by newly recruited activated macrophages. This walling off ofthe infected macrophages results in the characteristic granuloma. Thegranuloma is a compact, organized collection of activated macrophages,including epithelioid and multinucleated giant cells; surrounded by Tlymphocytes, and later by fibroblasts and collagen, which aggregatearound the macrophage core.

[0008] Mycobacterial dormancy results in a disease stage termed latenttuberculosis. An individual with latent tuberculosis may later develop acase of reactivated tuberculosis, and in fact, the majority of thetuberculosis cases reported in the United States are the result ofreactivation of a mycobacterial infection and not an initial infection.(Am. Rev. Respir. Dis. 146:1623-1633, 1992). Reactivation of the M.tuberculosis bacilli usually occurs in the apex of the lung where largenumbers of tubercle bacilli cause necrosis of the small bronchi of thelung. The characteristic bloodstained sputum of tuberculosis resultsfrom the erosion of small blood vessels during this necrotic process.

[0009] Approximately one-third of the population worldwide has beenestimated to be latently infected with M. tuberculosis. (Sudre et al.,Bull. W.H.O. 70:149-159, 1992). Currently, the tuberculin skin test isthe only available diagnostic for those infected with M. tuberculosis.Unfortunately, no currently available test can specifically identifylatently infected individuals. The tuberculin test is only capable ofidentifying all individuals either exposed to the pathogen or vaccinatedagainst the pathogen. Due to the high number of latently infectedindividuals and the risk of reactivation of tuberculosis in thoseindividuals, diagnostics and therapeutics targeted to latenttuberculosis need to be developed. In addition, the development of an invitro granuloma model for the study of mycobacteria and for thedevelopment of tuberculosis drug and vaccine candidates would bedesirable.

SUMMARY OF THE DISCLOSURE

[0010] An in vitro model for tuberculosis latency is described incertain embodiments of this disclosure. In particular, an in vitrogranuloma model and methods for using the model are provided. In someembodiments, the in vitro granuloma model contains human peripheralblood mononuclear cells, autologous macrophages and mycobacteria. Insome embodiments these components are combined in a low-attachmentcontainer. In specific examples, the in vitro granuloma model furthercontains fibroblasts, for example, human lung fibroblasts.

[0011] Further embodiments are methods for using the in vitro granulomamodel to screen new or known compounds for their effects on granuloma,for instance to screen candidate tuberculosis drugs, to identifycandidate tuberculosis vaccines, and to analyze and characterize theprocess of granuloma formation and granuloma necrosis.

[0012] Also provided herein are immunological methods for detectinglatent tuberculosis infections. Such methods are based on detectingspecific bacterial antigens (or antibodies against these antigens) thatare present in a subject with tuberculosis only (or predominately)during latent infection. By way of example, one such latency-specificantigen is alpha-crystallin (Acr).

[0013] Further embodiments include an immunological assay for detectionof latent tuberculosis in a subject, which assay involves contacting abiological sample from the subject, wherein the sample is suspected ofcontaining a first latency-specific biding partner (LSBP) (such as alatency-specific antigen or an antibody thereto), with a second(corresponding) LSBP, and detecting binding between the first LSBP andthe corresponding LSBP. Binding between the first and second LSBPs isindicative of latent tuberculosis in the subject. Thus, in one examplewhere the first LSBP is a M. tuberculosis latency-specific antigen (forinstance, Acr or an immunogenic fragment thereof), the correspondingLSBP may be an antibody that is capable of binding to that antigen.Where the first LSBP is an antibody, the corresponding LSBP (to make aspecific binding pair) is an antigen.

[0014] Also provided are kits for the detection of latent tuberculosisin a subject, which kits include at least one LSBP (e.g., alatency-specific antigen or antibody thereto) and instructions forcarrying out an immunological assay to detect binding of the LSBP to acognate LSBP found in a biological sample.

[0015] Also provided are kits comprising one or more elements of an invitro granuloma model, for instance cell culture media and, optionally,low-attachment containers and/or instructions for growing in vitrogranulomas.

[0016] Further embodiments provide methods for eliciting an immuneresponse in a subject by administering to the subject an immunestimulatory amount of a M. tuberculosis latency-specific antigen (e.g.,Acr), or immunogenic fragment thereof. Compositions containing suchimmunostimulatory molecules, and kits for their administration, are alsoprovided.

[0017] The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures and sequencelisting.

BRIEF DESCRIPTION OF THE FIGURES

[0018]FIG. 1 is a series of micrographs of aerosol-infected guinea piglung granuloma tissue. FIG. 1A shows tissue that was stained withhematoxylin and eosin (H & E). FIG. 1B shows tissue that was stainedwith acid-fast stain; representative mycobacterium are indicated by thearrows. FIG. 1C shows tissue that was subjected to immunohistochemicalstaining using a polyclonal antibody against the Acr protein; the arrowsindicate two stained mycobacteria

[0019]FIG. 2 is a ribonuclease protection assay (RPA) blot. Lanes 1-5are various negative and positive controls, as indicated. Lanes 6-9represent hybridizations to mRNA from mycobacteria grown 5 or 7 dayseither aerobically or in the anoxic chamber. Acr mRNA was observed atall four time points while rpoB mRNA was only observed in aerobicallygrown cultures.

[0020]FIG. 3 is a RPA blot. Lanes 1 and 2 represent hybridizations tomRNA from mycobacteria extracted after 7- or 12-day incubations in thein vitro granuloma model. Both acr and rpoB mRNA ere observed at bothtime points, likely indicating that aerobic bacilli were present in thegranuloma. Lanes 3 and 4 are positive controls; acr mRNA and acr andrpoB DNA, respectively.

[0021]FIG. 4 is a pair of agarose gels, showing the results of RT-PCR.Acr transcript is clearly induced with longer infection in the in vivomodel (lanes 2, 3, and 4), and is even more strongly induced in anoxicculture (lane 5).

[0022] Key: Lane 1, Uninfected Guinea Pig Lung; lane 2, MTB-InfectedGuinea Pig Lung—3 weeks P.I; lane 3, MTB-Infected Guinea Pig Lung—5weeks P.I.; lane 4, MTB-Infected Guinea Pig Lung—10 weeks P.I.; lane 5,MTB-7 Day Anoxic Culture.

[0023]FIG. 5 is a schematic drawing of an example anoxic growth vessel.

[0024]FIG. 6 is a pair of Western blots demonstrating the production ofthe N-terminal construct Acr-N-FLAG in transformant 3. The blot in FIG.6A was probed with anti-Acr polyclonal immunoglobin. The blot in FIG. 6Bwas probed with anti-FLAG epitope immunoglobin.

[0025]FIG. 7 is a pair of Western blots of whole cell lysatesdemonstrating the production of the C-terminal construct Acr-C-FLAG intwo Myobacterium smegmatis transformed strains versus non-transformed M.smegmatis and M. tuberculosis controls. The blot in FIG. 7A was probedwith anti-FLAG immunoglobin. The blot in FIG. 7B was probed withanti-Acr immunoglobin.

[0026]FIG. 8 shows five strips cut from a Western blot of whole celllysates; the arrows indicate the location of recombinant Acr protein.The strips were developed with the indicated primary antibodies. Inaddition to the control antibody, this blot demonstrates the presence oftyrosine phosphorylation in the final strip.

[0027]FIG. 9 shows two Western blots from culture supernatants of M.tuberculosis bacilli grown under various conditions. FIG. 9A was probedusing rabbit anti-Acr antibody; FIG. 9B is a control blot. Protein isdetected in 7 day and 12 month anoxic cultures and in vitro granuloma.Lower molecular weight variants are observed in the 12-month and invitro granuloma supernatants.

[0028] Key: Lane 1, molecular weight marker; lane 2, 3 day aerobicgrowth; lane 3, 7 day aerobic growth; lane 4, 7 day anoxic growth; lane5, blank; lane 6, 7 day react; lane 7, 12 months react; lane 8, controlcells; lane 9, 7 day in vitro granulomas; lane 10, M. tuberculosislysate.

[0029]FIG. 10 is a two-dimensional gel electrophoresis analysis of asample taken from M. tuberculosis grown under anoxic conditions. Acrprotein is indicated by the circle.

[0030]FIG. 11 is a Coomassie stained SDS-PAGE gel of culturesupernatants from M. tuberculosis bacilli cultured under a variety ofconditions.

[0031] Key: Lane 1, MW marker, lane 2, 5-day aerobic (logarithmic); lane3, anoxic 12 months; lane 4, anoxic 7 days; lane 5, 30-hour aerobicreactivated (logarithmic); lane 6, MW marker.

[0032]FIG. 12 is a graph showing the growth (measured by optical densityat 580 nm) of M. tuberculosis under anoxic and aerobic growthconditions.

SEQUENCE LISTING

[0033] The nucleic and amino acid sequences listed in the accompanyingsequence listing are shown using standard letter abbreviations fornucleotide bases, and three letter code for amino acids, as defined in37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown,but the complementary strand is understood as included by any referenceto the displayed strand. In the accompanying sequence listing:

[0034] SEQ ID NOs: 1 and 2 are the sequences of primers used to generatethe N-terminal FLAG-Acr fusion.

[0035] SEQ ID NOs: 3 and 4 are the sequences of primers used to generatethe C-terminal Acr-FLAG fusion.

DETAILED DESCRIPTION

[0036] I. Abbreviations Acr alpha (α) crystallin ELISA enzyme-linkedimmunosorbent assay HS human serum LSA latency-specific antigen LSBPlatency-specific binding partner PBMCs peripheral blood mononuclearcells RPA ribonuclease protection assay RT-PCR reverse-transcriptionpolymerase chain reaction

[0037] II. Terms

[0038] Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

[0039] In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

[0040] The terms “a,” “an,” and “the” as used herein are defined to meanone or more and include the plural unless the context is inappropriate.

[0041] The term “antibody” refers to a protein (or protein complex) thatincludes of one or more polypeptides substantially encoded byimmunoglobulin genes or fragments of immunoglobulin genes. Therecognized immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD and IgE, respectively.

[0042] The basic immunoglobulin (antibody) structural unit is generallya tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms “variable light chain”(V_(L)) and “variable heavy chain” (V_(H)) refer, respectively, to theselight and heavy chains.

[0043] As used herein, the term antibodies includes intactimmunoglobulins as well as a number of well-characterized fragmentsproduced by digestion with various peptidases, or genetically engineered“artificial” antibodies. Thus, for example, pepsin digests an antibodybelow the disulfide linkages in the hinge region to produce F(ab)′₂, adimer of Fab which itself is a light chain joined to V_(H)—C_(H) 1 by adisulfide bond. The F(ab)′₂ may be reduced under mild conditions tobreak the disulfide linkage in the hinge region thereby converting theF(ab)′₂ dimer into an Fab′ monomer. The Fab′ monomer is essentially aFab with part of the hinge region (see, Fundamental Immunology, W., E.Paul, ed., Raven Press, N.Y., 1993). While various antibody fragmentsare defined in terms of the digestion of an intact antibody, it will beappreciated that Fab′ fragments may be synthesized de novo eitherchemically or by utilizing recombinant DNA methodology. Thus, the termantibody as used herein also includes antibody fragments either producedby the modification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies.

[0044] Antibodies for use in the methods and devices of this disclosurecan be monoclonal or polyclonal. Merely by way of example, monoclonalantibodies can be prepared from murine hybridomas according to theclassical method of Kohler and Milstein (Nature 256:495-497, 1975) orderivative methods thereof. Detailed procedures for monoclonal antibodyproduction are described in Harlow and Lane (Antibodies, A LaboratoryManual, CSHL, New York, 1988).

[0045] The term “antigen” refers to a molecule, or fragment thereof,which can induce an immune response in a mammal. The term includesimmunogens and regions responsible for antigenicity or antigenicdeterminants. A chemical or biochemical structure, determinant, antigenor portion thereof that is capable of inducing the formation of anantibody can be referred to as being “antigenic.” “Antigenicdeterminant” refers to a region of a specified protein that isrecognized by an antibody.

[0046] When referring to macrophages, the term “autologous” refers tomacrophages that are derived from the same individual as the peripheralblood mononuclear cells. Alternatively, a macrophage cell line such as,but not limited to the THP-1 macrophage cell line is used as themacrophage component of the granuloma model. In one embodiment of thepresent disclosure, the beginning concentration of the autologousmacrophages is between approximately 5×10⁴ and 1×10⁶, per two-millilitersample, and optionally at a beginning concentration of approximately1×10⁶.

[0047] A “biological sample” is a sample of bodily fluid or tissue usedfor laboratory testing or examination. As used herein, biologicalsamples include all clinical samples useful for detection of microbialinfection in subjects.

[0048] Tissue samples may be taken from the oropharyngeal tract, forinstance from lung or bronchial tissue. Samples can be taken by biopsy(such as during a bronchoscopy) or during autopsy examination, asappropriate. Biological fluids include blood, derivatives, and fractionsof blood such as serum, urine, semen and fluids of the oropharyngealtract, such as sputum.

[0049] Examples of specimens for use with the current disclosure for thedetection of latent M. tuberculosis include conventional clinicalsamples, for instance blood or blood-fractions (e.g., serum), urine,bronchoalveolar lavage (BAL), sputum, and induced sputum samples.Techniques for acquisition of such samples are well known in the art.Blood and blood fractions can be prepared in traditional ways.Oropharyngeal tract fluids can be acquired through conventionaltechniques, including sputum induction, bronchoalveolar lavage (BAL),and oral washing. Obtaining a sample form oral washing involves havingthe subject gargle with an amount normal saline for about 10-30 secondsand then expectorate the wash into a sample cup.

[0050] The “condition” or “conditions” under which a DNA strand issynthesized include the presence of nucleotides, cations, andappropriate buffering agents in amounts and at temperatures such thatthe nucleic acid molecule and a DNA primer will anneal andoligonucleotides will be incorporated into a synthesized DNA strand.

[0051] As used herein, the terms “detecting” or “detection” refers toquantitatively or qualitatively determining the presence of abiomolecule under investigation.

[0052] “Epitope tags” are short stretches of amino acids to which aspecific antibody can be raised, which in some embodiments allow one tospecifically identify and track a protein tagged with the epitope.Detection of the tagged molecule can be achieved using a number ofdifferent techniques. Examples of such techniques include:immunohistochemistry, immunoprecipitation, flow cytometry,immunofluorescence microscopy, ELISA, immunoblotting (“western”), andaffinity chromatography. Examples of epitope tags include FLAG, T7, HA(hemagglutinin) and myc.

[0053] As used herein, the term “granuloma” refers to a compact,organized collection of activated macrophages, including epithelioid andmultinucleated giant cells, surrounded by T lymphocytes, fibroblasts andcollagen. It is to be understood, however, that the term “in vitrogranuloma” is not limited to a collection of cells as described above.The term “in vitro granuloma” refers to a collection or aggregate ofcells containing at least human peripheral blood mononuclear cells andautologous macrophages, wherein the collection or aggregate of cellsmimics the granuloma as described above. Whether the in vitro granulomamimics the granuloma as described above, and as found in vivo, isdetermined by methods known to those skilled in the art, such asmicroscopic examination of the cell aggregates, phenotypic analysis ofcells within the aggregates, via FACS (fluorescence activated cellsorter) analysis for example, and analysis of cytokine production by thecells within the aggregates.

[0054] As used herein, the term “human peripheral blood mononuclearcells” (PBMCs) includes, but is not limited to, monocytes, Blymphocytes, and T lymphocytes. The human PBMCs included in examples ofthe in vitro granuloma model can be monocytes and T lymphocytes.Optionally, in certain embodiments the in vitro granuloma model containsmonocytes at a beginning concentration of between approximately 5×10⁴and 1×10⁶, per two milliliter sample, and T lymphocytes at a beginningconcentration of between approximately 1×10⁵ and 1×10⁶. In someembodiments, the in vitro granuloma model contains monocytes at abeginning concentration of approximately 1×10⁶ and T lymphocytes at abeginning concentration of approximately 1×10⁶. It is to be understoodthat the term “beginning concentration” refers to the concentration ofmaterial as it is added to a low attachment container.

[0055] In some embodiments, the In vitro granuloma model containsfibroblasts, such as human lung fibroblasts. Optionally, the fibroblastscan be added at a beginning concentration of between approximately 1×10⁵and 1×10⁶, for instance, at a beginning concentration of approximately5×10⁵.

[0056] A “low attachment container” is a container whose surfaceinhibits or reduces the attachment of cells in culture. In someembodiments, the low attachment container is a low attachment tissueculture dish such as, but not limited to, the COSTAR™ Ultra LowAttachment Surface or Clusters (Costar Corp., Cambridge, Mass.) used inaccordance with the manufacturer's recommended procedures. Optionally,the low attachment container can have a surface composed of a covalentlybound hydrogel layer that is hydrophilic and neutrally charged, so thatit inhibits (for instance, by 5%, 10%, 20%, 40%, 50% or more compared toa non-coded container) the attachment and activation of macrophages andneutrophils. Because proteins and other biomolecules passively adsorb tosurfaces through hydrophobic and ionic interactions, a hydrogel surfacenaturally inhibits non-specific immobilization via these forces, thusinhibiting subsequent cell attachment. Optionally, the surface of thelow attachment container may be rehydrated at a temperature consistentwith the application or cell growth requirements of the cells describedabove and the rehydration media aspirated or decanted prior to theaddition of cells and fresh media. Alternatively, the cells may be addeddirectly to the rehydration media

[0057] “In vitro amplification” refers to techniques that increase thenumber of copies of a nucleic acid molecule in a sample or specimen. Anexample of in vitro amplification is the polymerase chain reaction(PCR), in which a biological sample collected from a subject iscontacted with a pair of oligonucleotide primers, under conditions thatallow for the hybridization of the primers to a nucleic acid template inthe sample. The primers are extended under suitable conditions,dissociated from the template, and then re-annealed, extended, anddissociated to amplify copies of the nucleic acid. Other examples of invitro amplification techniques include strand displacement amplification(see U.S. Pat. No. 5,744,311); transcription-free isothermalamplification (see U.S. Pat. No. 6,033,881); repair chain reactionamplification (see WO 90/01069); ligase chain reaction amplification(see EP-A-320 308); gap filing ligase chain reaction amplification (seeU.S. Pat. No. 5,427,930); coupled ligase detection and PCR (see U.S.Pat. No. 6,027,889); and NASBA™ RNA transcription-free amplification(see U.S. Pat. No. 6,025,134). The product of in vitro amplification maybe characterized by electrophoresis, restriction endonuclease cleavagepatterns, oligonucleotide hybridization or ligation, and/or nucleic acidsequencing, using standard techniques.

[0058] An “isolated” biological component (such as a nucleic acidmolecule, protein or organelle) has been substantially separated orpurified away from other biological components in the cell of theorganism in which the component naturally occurs, i.e., otherchromosomal and extra-chromosomal DNA and RNA, proteins and/ororganelles. Nucleic acids and proteins that have been “isolated” includenucleic acids and proteins purified by standard purification methods.The term also embraces nucleic acids and proteins prepared byrecombinant expression in a host cell, as well as chemically synthesizednucleic acids. As with the term purified, isolated is a relative term.

[0059] A “label” is any molecule or composition that is detectable by,for instance, spectroscopic, photochemical, biochemical, immunochemical,electrical, optical, or chemical means. Examples of labels, includingradioactive isotopes, enzyme substrates, co-factors, ligands,chemiluminescent or fluorescent agents, haptens, enzymes, colloidal goldparticles, colored latex particles, and epitope tags, have beendisclosed previously and are known to those of ordinary skill (see, forinstance, U.S. Pat. Nos. 4,275,149; 4,313,734; 4,373,932; and4,954,452).

[0060] The attachment of a compound (e.g., an antibody) to a label canbe through covalent bonds, adsorption processes, hydrophobic and/orelectrostatic bonds, as in chelates and the like, or combinations ofthese bonds and interactions and/or may involve a linking group.

[0061] “Latent tuberculosis” refers to a stage in the M. tuberculosisinfection where the bacilli remain viable but are slowly replicating ornon-replicating, may be encapsulated in localized lesions within thelung, and do not cause active necrotic disease. The latent stage mayexist for the remainder of a host's life, or the infection mayreactivate during, for instance, a period of decreased host immunity orin response to other stressors. Though latent M. tuberculosis infectionshave not previously been able to be specifically identified, they arewithin the group of individuals that possess a positive tuberculin skintest but do not possess the characteristic symptoms of active disease.

[0062] A “latency specific antigen” is an antigen that is expressed athigher levels (or exclusively) by a M. tuberculosis bacterium in itsdormant or stationary rather than its active or logarithmic phase ofgrowth. Latency specific antigens (LSAs) can be identified, forinstance, by comparing the protein expression found in in vitro culturedM. tuberculosis grown under standard aerobic/logarithmic conditions withbacilli grown under conditions that mimic latency (e.g., in a latencymodel).

[0063] A “linking group” is a chemical arm between two compounds, forinstance a compound and a label (e.g., an antibody and a label). Toaccomplish the requisite chemical structure of the linkage, each of thereactants must contain a reactive group. Representative combinations ofsuch groups are amino with carboxyl to form amide linkages; carboxy withhydroxy to form ester linkages; amino with alkyl halides to formalkylamino linkages; thiols with thiols to form disulfides; or thiolswith maleimides or alkylhalides to form thioethers. Hydroxyl,carboxylamino and other functionalities, where not present in the nativecompound, may be introduced by known methods.

[0064] Likewise, a wide variety of liking groups may be employed. Thestructure of the linkage should be a stable covalent linkage formed toattach two compounds to each other (e.g., the label to the antibody). Insome cases the linking group may be designed to be either hydrophilic orhydrophobic in order to enhance a desired characteristic, for instance abinding characteristic of a modified ligand and its cognate receptor.The covalent linkages should be stable relative to the solutionconditions to which linked compounds are subjected.

[0065] Examples of linking groups will be from 1-20 carbons and 0-10heteroatoms (NH, O, S) and may be branched or straight chain. Withoutlimiting the foregoing, it should be obvious that only combinations ofatoms that are chemically compatible comprise the lining group. Forexample, amide, ester, thioether, thiol ester, keto, hydroxylcarboxyl,and ether groups in combinations with carbon-carbon bonds are particularexamples of chemically compatible linking groups.

[0066] The term “mycobacteria” as used herein includes, but is notlimited to, M. tuberculosis. Any mycobacteria that form granulomas canbe used in the compositions and methods provided herein. Exemplarymycobacteria include M. avium, M. bovis, M. marinum, M. ulcerans, M.smegmatis, and M. haemophilum. Optionally, the beginning concentrationof mycobacteria in the in vitro granuloma model is between approximately1×10¹ and 1×10⁵ cfu/2 ml sample.

[0067] A first nucleic acid sequence is “operably linked” with a secondnucleic acid sequence when the first nucleic acid sequence is placed ina functional relationship with the second nucleic acid sequence. Forinstance, a promoter is operably linked to a coding sequence if thepromoter affects the transcription or expression of the coding sequence.Generally, operably lined DNA sequences are contiguous and, wherenecessary to join two protein-coding regions, in the same reading frame.

[0068] “Peptides,” “polypeptides,” and “oligopeptides” are chains ofamino acids (typically L-amino acids) whose alpha carbons are linkedthrough peptide bonds formed by a condensation reaction between thecarboxyl group of the alpha carbon of one amino acid and the amino groupof the alpha carbon of another amino acid. The terminal amino acid atone end of the chain (i.e., the amino terminal) has a free amino group,while the terminal amino acid at the other end of the chain (i.e., thecarboxy terminal) has a free carboxyl group. As such, the term “aminoterminus” (abbreviated N-terminus) refers to the free alpha-amino groupon the amino acid at the amino terminal end of the peptide, or to thealpha amino group (imino group when participating in a peptide bond) ofan amino acid at any other location within the peptide. The term“carboxy terminus” (abbreviated C-terminus) refers to the free carboxylgroup on the amino acid at the carboxy terminal end of a peptide, or tothe carboxyl group of an amino acid at any other location within thepeptide.

[0069] Typically, the amino acids making up a peptide are numbered inorder, starting at the amino terminus and increasing in the directiontoward the carboxy terminus of the peptide. Thus, when one amino acid issaid to “follow” another, that amino acid is positioned closer to thecarboxy terminal end of the peptide than the preceding amino acid.

[0070] As used herein, the term “primer” or “DNA primer” means anoligonucleotide that anneals to a nucleic acid molecule in a particularorientation to allow for the synthesis of a nascent DNA strand.

[0071] As used herein, the phrase “primer pair” refers to two primers,one having a forward designation and the other having a reversedesignation (relative to their respective orientations when annealed toa double-stranded DNA molecule that consists of a sense and antisensesequence). Under in vitro amplification conditions, the forward primeranneals to and primes amplification of the sense sequence and thereverse primer anneals to and primes amplification of the antisensesequence. Primers can be selected for use in an amplification reactionon the basis, for instance, of having minimal complementarity with otherprimers in the reaction (to minimize the formation of primer dimers) andhaving T_(m) values with a range of reaction temperatures appropriatefor the amplification method, such as PCR. In addition, primers can beselected to anneal with specific regions of a DNA or RNA template suchthat the resulting DNA amplification product of specific size, forinstance from 100 to 5000 base pairs in length, for instance around 300base pairs in length or longer.

[0072] By “probe” is meant a nucleic acid sequence that can be used forselective hybridization with complementary nucleic acid sequences fortheir detection. The probe varies in length, for instance from about 5to 100 nucleotides, or from about 10 to 50 nucleotides, or about 18 to24 nucleotides. A “labeled probe” comprises an isolated nucleic acidprobe attached to a detectable label or other reporter molecule. Methodsfor labeling and guidance in the choice of labels appropriate forvarious purposes are discussed, e.g., in Sambrook et. al. (In MolecularCloning: A Laboratory Manual, CSHL, New York, 1989) and Ausubel et al.(In Current Protocols in Molecular Biology, John Wiley & Sons, New York,1998).

[0073] A “promoter” includes one or more nucleic acid sequences thatdirect transcription of a nucleic acid. A promoter includes nucleic acidsequences near the start site of transcription, such as, in the case ofa polymerase II type promoter, a TATA element. A promoter may alsoinclude distal enhancer or repressor elements that can be located asmuch as several thousand base pairs from the start site oftranscription.

[0074] The term “purified” as it is used herein does not requireabsolute purity; rather, it is intended as a relative term. Thus, forexample, a purified nucleic acid (or protein or other compound)preparation is one in which the specified molecule (or type of molecule)is more enriched than it is in its generative environment, for instancewithin a cell or in a biochemical reaction chamber (as appropriate). Apreparation of a “substantially pure” substance, for instance asubstantially pure nucleic acid, may be purified such that the desirednucleic acid represents at least 50% of the total nucleic acid contentof the preparation. In certain embodiments, a substantially purepreparation will represent at least 60%, at least 70%, at least 80%, atleast 85%, at least 90%, or at least 95% or more desired molecule in thepreparation.

[0075] A “recombinant” nucleic acid is one that has a sequence that isnot naturally occurring or has a sequence that is made by an artificialcombination of two otherwise separated segments of sequence. Thisartificial combination can be accomplished by chemical synthesis or,more commonly, by the artificial manipulation of isolated segments ofnucleic acids, e.g., by genetic engineering techniques.

[0076] The term “residue” is used herein to refer to an amino acid (D orL), or an amino acid mimetic, that is incorporated into a peptide by anamide bond. As such, the amino acid may be a naturally occurring aminoacid or, unless otherwise limited, may encompass analogs of naturalamino acids that function in a manner similar to the naturally occurringamino acids (i.e., amino acid mimetics). Moreover, an amide bond mimeticincludes peptide backbone modifications well known to those of ordinaryskill in the art.

[0077] The phrase “sequence identity” refers to the similarity betweentwo nucleic acid sequences, or two amino acid sequences, and isexpressed in terms of the similarity between the sequences. Sequenceidentity is frequently measured in terms of percentage identity (orsimilarity or homology); the higher the percentage, the more similar thetwo sequences are.

[0078] Methods of alignment of sequences for comparison are well knownin the art. Various programs and alignment algorithms are described in:Smith & Waterman Adv. Appt Math. 2: 482, 1981; Needleman & Wunsch J.Mol. Biol. 48:443, 1970; Pearson & Lipman Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp Gene, 73: 237-244, 1988; Higgins & SharpCABIOS 5: 151-153, 1989; Corpet et al. Nuc. Acids Res. 16, 10881-90,1988; Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992;and Pearson et al. Meth. Mol. Bio. 24, 307-31, 1994. Altschul et al. (J.Mol. Biol. 215:403-410, 1990), presents a detailed consideration ofsequence alignment methods and homology calculations.

[0079] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul etal., J. Mol. Biol. 215:403-410, 1990) is available from several sources,including the National Center for Biotechnology Information (NCBI,Bethesda, Md.) and on the Internet, for use in connection with thesequence analysis programs blastp, blastn, blastx, tblastn and tblastx.

[0080] An alternative indication that two nucleic acid molecules areclosely related is that the two molecules hybridize to each other understringent conditions. Stringent conditions are sequence-dependent andare different under different environmental parameters. Generally,stringent conditions are selected to be about 5° C. to 20° C. lower thanthe thermal melting point (Tm) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence remains hybridizedto a perfectly matched probe or complementary strand. Conditions fornucleic acid hybridization and calculation of stringencies can be foundin Sambrook et al (In Molecular Cloning: A Laboratory Manual, CSHL, NewYork, 1989) and Tijssen (Laboratory Techniques in Biochemistry andMolecular Biology—Hybridization with Nucleic Acid Probes Part I, Chapter2, Elsevier, New York, 1993). Nucleic acid molecules that hybridizeunder stringent conditions to a target sequence will typically hybridizeto a probe based on either an entire target protein encoding sequence,or selected portions of the encoding sequence, under wash conditions of2×SSC at 50° C.

[0081] Nucleic acid sequences that do not show a high degree of identitymay nevertheless encode similar amino acid sequences, due to thedegeneracy of the genetic code. It is understood that changes in nucleicacid sequence can be made using this degeneracy to produce multiplenucleic acid molecules that all encode substantially the same protein orthe identical protein.

[0082] Furthermore, one of ordinary skill in the art will recognize thatindividual substitutions, deletions or additions in the amino acidsequence of the protein, or in the nucleotide sequence encoding for theamino acids in the protein, which alter, add or delete a single aminoacid or a small percentage of amino acids (in some instances less than5%, or even less than 1%) in an encoded sequence are conservativelymodified variations, wherein the alterations result in the substitutionof an amino acid with a chemically similar amino acid, and so long asthe resultant variant still retains a substantial proportion of aproperty or activity, such as an immunostimulatory property (e.g., aprotective immune response in a subject), of the base protein.Envisioned in specific embodiments are molecules in which there is nomore than one amino acid substitution, no more than about threesubstitutions, or about 5, 10, or even 20 substitutions, so long as theresultant variant retains a substantial proportion (e.g., at least 20%,at least 30%, at least 50%, at least 75%, at least 80%, at least 90%, atleast 95%, at least 98%, or more) of an immunostimulatory or otherproperty of the base protein. Some variant embodiments are expected tohave greater immunostimulatory properties than the protein or peptidefrom which they are derived.

[0083] Conservative amino acid substitution tables providingfunctionally similar amino acids are well known to one of ordinary skillin the art. The following six groups are examples of amino acids thatare considered to be conservative substitutions for one another:

[0084] 1) Alanine (A), Serine (S), Threonine (T);

[0085] 2) Aspartic acid (D), Glutamic acid (E);

[0086] 3) Asparagine (N), Glutamine (Q);

[0087] 4) Arginine (R), Lysine (K);

[0088] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0089] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0090] The term “specific binding agent” as used herein refers to anagent that binds substantially only to a defined target. Thus aprotein-specific binding agent binds substantially only the specifiedprotein. The term “protein specific binding agent” includes anti-proteinantibodies (and functional fragments thereof) and other agents (such assoluble receptors) that bind substantially only to the specifiedprotein.

[0091] Anti-protein antibodies (such as anti-Acr antibodies) may beproduced using standard procedures described in a number of texts,including Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, NewYork, 1988). The determination that a particular agent bindssubstantially only to the specified protein, or component epitopesthereof may readily be made by using or adapting routine procedures. Onesuitable in vitro assay makes use of the Western blotting procedure(described in many standard texts, including Harlow and Lane(Antibodies, A Laboratory Manual, CSHL, New York, 1988)). Westernblotting may be used to determine that a given protein binding agent,such as an anti-Acr monoclonal antibody, binds substantially only to thespecified protein.

[0092] Shorter fragments of antibodies can also serve as specificbinding agents. For instance, Fabs, Fvs, and single-chain Fvs (SCFvs)that bind to Acr would be Acr-specific binding agents. These antibodyfragments are defined as follows: (1) Fab, the fragment which contains amonovalent antigen-binding fragment of an antibody molecule produced bydigestion of whole antibody with the enzyme papain to yield an intactlight chain and a portion of one heavy chain; (2) Fab′, the fragment ofan antibody molecule obtained by treating whole antibody with pepsin,followed by reduction, to yield an intact light chain and a portion ofthe heavy chain; two Fab′ fragments are obtained per antibody molecule;(3) (Fab′)₂, the fragment of the antibody obtained by treating wholeantibody with the enzyme pepsin without subsequent reduction; (4)F(ab′)₂, a dimer of two Fab′ fragments held together by two disulfidebonds; (5) Fv, a genetically engineered fragment containing the variableregion of the light chain and the variable region of the heavy chainexpressed as two chains; and (6) single chain antibody (“SCA”), agenetically engineered molecule containing the variable region of thelight chain, the variable region of the heavy chain, linked by asuitable polypeptide linker as a genetically fused single chainmolecule. Methods of making these fragments are routine.

[0093] A “specific binding partner” is a member of a pair of molecules(the “specific binding pair”) capable of recognizing and binding to astructural aspect of another molecule by means of specific, noncovalentinteractions that depend on the three-dimensional structures of themolecules involved. Typical pairs of specific binding partners includeantigen/antibody, hapten/antibody, hormone/receptor, nucleic acidstrand/complementary nucleic acid strand, substrate/enzyme,inhibitor/enzyme, apoprotein/cofactor, carbohydrate/lectin,biotin/(strept)avidin, and virus/cellular receptor.

[0094] A specific binding pair that includes at least one immunologicalmolecule (such as an antibody or antigen) can be referred to as aspecific immunological binding pair, and the immunological molecule(s)as specific immunological binding partner(s).

[0095] An example of a specific binding pair is a latency-specificbinding pair, which includes a molecule that is a latency-specificmolecule (such as a latency-specific antigen) and a molecule that is aspecific binding partner for that latency-specific molecule.

[0096] The phrase “specifically binds to an analyte” or “specificallyimmunoreactive with,” when referring to an antibody, refers to a bindingreaction or interaction which is determinative of the presence of theanalyte or epitope in a heterogeneous population of molecules such asproteins and other biological molecules. Thus, under designatedimmunoassay conditions, specified antibodies bind to a particularanalyte or epitope and do not bind in a significant amount to otheranalytes or epitope present in the sample. A variety of immunoassayformats may be used to select antibodies specifically immunoreactivewith a particular analyte or epitope. For example, solid-phase ELISAimmunoassays are routinely used to select monoclonal antibodiesspecifically immunoreactive with a protein. See Harlow and Lane,Antibodies, A Laboratory Manual, CSHP, New York (1988), for adescription of immunoassay formats and conditions that can be used todetermine specific immunoreactivity.

[0097] The term “subject” as used herein refers to living multi-cellularvertebrate organisms, a category that includes both human and non-humanmammals. The term “subject” includes both human and veterinary subjects.

[0098] When referring to cytokines or other biological materials, theterm “steady state level” refers to the level of the cytokine orbiological material produced in uninfected cells.

[0099] The term “synthetic polypeptide” refers to a polypeptide formed,in vitro, by joining amino acids in a particular order, using the toolsof organic chemistry to form the peptide bonds.

[0100] A “transformed” cell is a cell into which has been introduced anucleic acid molecule by molecular biology techniques. As used herein,the term transformation encompasses all techniques by which a nucleicacid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of naked DNA by electroporation, lipofection, andparticle gun acceleration.

[0101] The term “vaccine” is used herein to mean a composition usefulfor stimulating a specific immune response in a vertebrate.

[0102] The term “vector” as used herein refers to a nucleic acidmolecule as introduced into a host cell thereby producing a transformedhost cell. A vector may include nucleic acid sequences that permit it toreplicate in a host cell, such as an origin of replication. A vector mayalso include one or more selectable marker genes and other geneticelements known in the art.

[0103] Unless otherwise explained, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this disclosure belongs. It isfurther to be understood that all base sizes or amino acid sizes, andall molecular weight or molecular mass values, given for nucleic acidsor polypeptides are approximate, and are provided for description.Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present disclosure,suitable methods and materials are described below. In case of conflict,the present specification, including explanations of terms, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

[0104] III. Description of Several Embodiments

[0105] Provided herein in a first embodiment is an immunological assaymethod for detection of latent tuberculosis in a subject. Such methodsinclude contacting a biological sample that may contain a firstlatency-specific binding partner (LSBP) from the subject with acorresponding LSBP, and detecting binding between the first LSBP and thecorresponding LSBP, wherein such binding is indicative of latenttuberculosis in the subject. In some specific examples of these methods,the first LSBP is an antibody, and the corresponding LSBP is alatency-specific M. tuberculosis antigen (for instance, α-crystallin(Acr) or an immunogenic fragment thereof). In other specific examples ofthe methods, the first LSBP is a latency-specific M. tuberculosisantigen, and the corresponding LSBP is an antibody. In certain specificexamples of the methods, the antigen is Acr or an immunogenic fragmentthereof.

[0106] Another embodiment is a kit for the detection of latenttuberculosis in a subject. Such kits include at least one LSBP andinstructions for carrying out an immunological assay method fordetection of latent tuberculosis in a subject.

[0107] This disclosure also provides a method of eliciting an immuneresponse in a subject. The methods include introducing into the subjectan immune stimulatory amount of a M. tuberculosis latency-specificantigen or immunogenic fragment thereof, or a nucleic acid moleculeencoding such an antigen (eg., Acr) or immunogenic fragment thereof. Incertain specific examples, the method is a method of inhibiting ortreating a latent tuberculosis infection in the subject. In particularexamples of the methods, the elicited immune response results indecreased susceptibility of the subject to latent infection by M.tuberculosis.

[0108] This disclosure further provides a kit for eliciting an immuneresponse in a subject. Such kits include an immune stimulatory amount ofa M. tuberculosis latency-specific antigen or immunogenic fragmentthereof, or a nucleic acid molecule encoding such an antigen orimmunogenic fragment, and instructions for carrying out a method ofeliciting an immune response in a subject. Particular examples of thekit include instructions for administering a component of the kit to apatient with a possible latent tuberculosis infection.

[0109] Further embodiments provide in vitro granulomas that includeperipheral blood mononuclear cells, autologous macrophages, andmycobacteria. In such granulomas, the peripheral blood mononuclear cellsare human peripheral blood mononuclear cells selected from the groupconsisting of monocytes, B lymphocytes, T lymphocytes, and combinationsthereof. In certain examples of the granuloma, the mycobacteria are M.tuberculosis. Certain examples of the granuloma further includefibroblasts.

[0110] Also provided is a method for producing an in vitro granuloma,which method involves combining peripheral blood mononuclear cells,autologous macrophages, and mycobacteria in a low attachment containerand incubating the combination for a sufficient amount of time to formthe in vitro granuloma. In some, specific examples, fibroblasts areadded to the combination. In some specific examples, exogenouscytokine(s) are added to the container in sufficient amount to enhanceproduction of the in vitro granuloma. In some instances, the exogenouscytokine is IL-2, IFN-γ, TNF-α or a combination of two or more thereof.

[0111] Another embodiment provided herein is a method of screening atuberculosis drug candidate for anti-tuberculosis therapeutic activity.Such methods include combining the drug with an in vitro granulomacomprising peripheral blood mononuclear cells, autologous macrophages,and mycobacteria, and determining whether the drug inhibitsmycobacterial viability. In some specific examples, the peripheral bloodmononuclear cells are human peripheral blood mononuclear cells selectedfrom the group consisting of monocytes, B lymphocytes, T lymphocytes,and combinations thereof.

[0112] Still another embodiment provided herein is a method of screeninga tuberculosis drug candidate for anti-tuberculosis therapeutic activitythat includes combining the drug with an in vitro granuloma comprisingperipheral blood mononuclear cells, autologous macrophages, andinactivated mycobacteria, and determining whether the drug inhibitsreactivation of mycobacteria contained in the granuloma. In certainspecific examples of the methods, the mycobacteria are M. tuberculosis.

[0113] Also provided is a method of screening a tuberculosis vaccinecandidate that includes determining whether a mutant mycobacteria has areduced ability, when compared against a wild type mycobacteria, toinduce latency, survive, reactivate or induce granuloma necrosis in anin vitro granuloma comprising peripheral blood mononuclear cells,autologous macrophages, and the mutant mycobacteria. In some specificexamples of the methods, the in vitro granuloma further comprisesfibroblasts. In certain examples of the methods, the mutant mycobacteriaincludes a mycobacteria strain having a mutation in a latency gene. Inother examples, the mutant mycobacteria is a Mycobacterium tuberculosisstain having a mutation in a gene selected from the group consisting ofacr, a sigma factor gene, oxyR and aphC. In particular examples, thesigma factor gene is selected from the group consisting of sigF, sigC,and sigH.

[0114] Additional embodiments are kits for producing an in vitrogranuloma, including a culture medium and instructions for carrying outa method of screening a tuberculosis vaccine candidate that includesdetermining whether a mutant mycobacteria has a reduced ability, whencompared against a wild type mycobacteria, to induce latency, survive,reactivate or induce granuloma necrosis in an in vitro granulomacomprising peripheral blood mononuclear cells, autologous macrophages,and the mutant mycobacteria. In certain examples, the kit furtherincludes a low attachment container and in certain specific examples,the kit further includes an amount of a cytokine.

[0115] IV. Production and use of the In Vitro Granuloma Model

[0116] This disclosure provides in this embodiment methods for producingand using an in vitro granuloma model, which model provides a consistentreplicable, and reliable laboratory system. The in vitro granuloma canbe used in many applications, for instance to study granuloma formationand maintenance, as well as to identify and characterize compounds thataffect granuloma formation, maintenance, or reversion, and to studyaspects of mycobacterial, including particularly mycobacterial latency.

[0117] In general, the in vitro granuloma is formed by combiningperipheral blood mononuclear cells with macrophages and mycobacteria.The mixture of cells is incubated for a number of days to encourageformation of aggregates in the cell culture. Formation of the granulomasis further encouraged by use of low attachment containers in certainembodiments. After formation of cell aggregates, fibroblasts canoptionally be added to the culture. In some embodiments, exogenouscytokines are added to the growth medium, for instance, IL-2, IFN-γ,and/or TNF-α, for instance prior to infection with mycobacteria.

[0118] By way of more specific example, autologous macrophages andmycobacteria are combined in one or more wells of a low attachmentcontainer, such as wells of a tissue culture dish treated to inhibit allattachment. Peripheral blood mononuclear cells (PBMCs), optionally 1×10⁶cells in a cell culture media (such as RPMI plus 10% human serum, HS(Lampire Biological Laboratories, Pipersville, Pa.)), are combined withthe macrophages and mycobacteria and incubated at a temperature at whichthe cells will grow for between 5 and 7 days. When aggregates reach adiameter of approximately 1 mm, fibroblasts (optionally human lungfibroblasts) may be added.

[0119] Secretion of a variety of chemoattractant cytokines followingphagocytosis of M. tuberculosis bacilli by the macrophage is importantnot only to the formation of the granuloma but also to its maintenance.Because of this, progression of the in vitro granuloma can be monitoredby measuring cytokine levels. For cytokine analysis, supernatants areharvested, filter sterilized, and assayed by a known technique such asELISA.

[0120] It is also beneficial in some embodiments to add exogenouscytokine to the medium, for instance during formation of the in vitrogranuloma; in some instances, addition of cytokine enhances aggregateformation. By way of example, IL-2 (at 10 units/ml, for instance), IFN-γ(at 2 ng/ml, for instance), or TNF-α (at 50 ng/ml, for instance)(Endogen, Woburn, Mass.) or combinations of two or three cytokines areadded to the cells prior to mycobacterial infection. When in vitrogranulomas were maintained for longer periods of time, for instance for9 days, cytokines could beneficially be added a second time. In somespecific examples, the same amounts and kinds of cytokines were added atday 5 after starting the cultures.

[0121] Similarly, gene expression can be used to characterize the invitro granuloma For RT-PCR or RPA analysis, aggregates are collected,washed and the RNA extracted. For histopathology, cells are fixed in afixative, such as 10% formalin, and processed as tissue.

[0122] The in vitro granuloma model of the present disclosure has avariety of uses. For example, the in vitro granuloma model can be usedto analyze and characterize the process of granuloma formation andgranuloma necrosis. It can also be also used to characterize M.tuberculosis genes that are differentially expressed when themycobacterium is located inside a granuloma versus when themycobacterium in not located inside a granuloma.

[0123] The model, for instance when employed without the addition ofexogenous cytokines, is useful to characterize native cytokineproduction by the cells composing a granuloma. In one embodiment, thecytokine levels produced by the in vitro granuloma model are compared tothe steady state levels of the cytokines. The granuloma model is alsouseful for analyzing mycobacterial viability, as well as forpathophysiologic analyses.

[0124] V. Drug and Immune-Stimulating Compound Candidate Screening

[0125] The in vitro granuloma model described herein can be used foraccurately screening candidate compounds, for instance, tuberculosisdrug candidates. Methods of screening a tuberculosis drug candidateinclude adding the drug candidate to the in vitro granuloma model anddetermining whether the drug kills mycobacteria contained in thegranuloma or otherwise alters the physiology of the bacilli or othercells in the granuloma. The model is optionally used to screen drugs foruse in treating latent mycobacterial infections.

[0126] The in vitro granuloma model is also useful for screeningimmunostimulatory compounds, for example tuberculosis vaccinecandidates. By way of example, the in vitro granuloma model is used as aprecursor to animal studies, for instance to test mutant stains of M.tuberculosis that may be used as vaccines to prevent or reduce M.tuberculosis infection or the reactivation of latent mycobacterialinfections. Animal study precursors help to reduce the costs andnegative connotations associated with animal experimentation. Examplesof the in vitro granuloma model provided herein are less expensive andmuch more rapid than conventional vaccine screening tests that rely ondata generated from animal experiments.

[0127] A mutant M. tuberculosis strain can be constructed by methodsknown to those of skill in the art, for instance by inducing orscreening for a mutation, such as a mutation in one of the latency genesselected from, but not limited to, acr, sigF, sigC, sigH, and othersigma factors, oxyR and aphC. Examples of methods known to those ofskill in the art include in vitro mutagenesis and in vivo mutagenesis.The mutation can be a deletion of all or part of one or more of theselatency genes or an insertion or substitution in one or more of theselatency genes. When referring to mycobacterial strains in general or anM. tuberculosis strain specifically, the terms “mutated” and “mutant”refer to strains having one or more mutations that inhibit or preventthe strain from shifting down into a dormant or latent state, or thatinhibit or prevent the strain from reactivating after shifting down intoa dormant or latent state. Dormancy is a state of slow or stoppedbacterial replication, but with some ongoing metabolism, whereasreactivation involves bacterial replication and log phase biochemistry.

[0128] In examples of methods for identifying mutated strains that canserve as vaccines, a mutated mycobacterial strain as described herein isadded to the in vitro granuloma model in place of a wild-typemycobacteria strain. The efficacy of the mutated mycobacteria strain isdetermined based upon a reduced ability of the mutated strain to induceformation of a latent mycobacterial infection state, survive within thegranuloma, reactivate from a latent state to an active state, and/or areduced ability of the mutant strain to induce granuloma necrosis ascompared to a wild-type mycobacterial strain. Cytokine production in thein vitro granuloma containing the mutated mycobacteria strain also canbe analyzed and compared to cytokine production in a control in vitrogranuloma model containing a wild-type mycobacteria strain.

[0129] VI. Identification of Latency-Specific Antigens

[0130] To gain insight into the molecular mechanisms of mycobacterialdormancy, and to provide molecules for detecting and/or tracking dormantinfection, genes and proteins suspected of being involved in theadaptation to anoxia have been investigated. Results usingreverse-transcription polymerase chain reaction (RT-PCR) technology (asdescribed herein) have confirmed differential expression of variousputative stress-response genes. Two of these genes that demonstrateincreased activity in the anaerobic (shift-down) granuloma model areinvolved in oxidative stress response, oxyR and aphC (Dhadayauthapani etal., J. Bacteriol. 178:3641-3649, 1996). The third encodes α-crystallin,an ATP-independent chaperon (Henriques et al., J. Bacter. 179:1887-1897,1997; Horwitz, Proc. Nat'l. Acad. Sci. USA 89:10449-10453, 1992),reported to be required for bacilli growth within macrophages (Yuan etal., Microbiol. 95, 16:9578-9583, 1998).

[0131] Latency-specific antigens in addition to those discussed inspecific embodiments disclosed herein can be identified based on theirpreferential expression by latent mycobacteria, particularly M.tuberculosis, in comparison to mycobacteria that are not in latentphase. By way of example, differential expression can be detected usingtwo dimensional gel electrophoresis of proteins extracted from latentand non-latent mycobacteria (e.g., bacteria cultured under anoxicconditions and aerobic conditions).

[0132] Alternatively, gene-chip (or cDNA microarray) analysis can beperformed to detect preferential mRNA expression in latent (e.g., invitro granuloma) versus non-latent (i.e., aerobic) cultures ofmycobacteria. For instance, subtractive hybridization can be carried outby spotting all of the transcripts expressed in one culture or theother. The chip can then be probed using labeled (e.g., with a cyaninedye) latent and labeled non-latent mRNA (or cDNA) pools, anddifferential expression detected using known techniques.

[0133] The prototypical latency-specific antigen is α-crystallin; itsidentification and characterization is described in more detail herein.

[0134] VII. Detection of Latent Tuberculosis

[0135] It has been surprisingly found that latent M. tuberculosis canproduce a high enough level of latency-specific antigens that suchantigens, and/or antibodies reactive therewith, can be detected insubjects with latent M. tuberculosis infection.

[0136] With the demonstration herein that latent tuberculosis organismsproduce specific antigens that elicit immunogenic responses in subjects,methods for the detection of latency-specific antigens, and/orantibodies to latency-specific antigens, and for the detection and/ordiagnosis of latent infections, are now enabled.

[0137] Latency-specific antigens, or antibodies that recognize anepitope of a M. tuberculosis latency-specific protein (such as Acr) canbe detected in samples from a subject, for instance serum or otherbiological fluid, using known immunological techniques. The presence ofsuch latency-specific antigens or antibodies (e.g., circulatingantibodies specific for an Acr epitope) indicates that the subjectsuffers from a latent tuberculosis infection.

[0138] Many techniques are commonly known in the art for the detectionand quantification of antigen. Most commonly, purified antigen will bebound to a substrate, the antibody of the sample will bind via its Fabportion to this antigen, the substrate will then be washed and a second,labeled antibody will then be added which will bind to the Fc portion ofthe antibody that is the subject of the assay. The second, labeledantibody will be species specific, i.e., if the serum is from a human,the second, labeled antibody will be anti-human-IgG antibody. Thesubstrate will then be washed and the amount of the second, labeledantibody that has been bound will be detected and quantified by standardmethods.

[0139] Examples of methods for the detection of antibodies in biologicalsamples, including methods employing dip strips or other immobilizedassay devices, are disclosed for instance in the following patents: U.S.Pat. No. 5,965,356 (Herpes simplex virus type specific seroassay); U.S.Pat. No. 6,114,179 (Method and test kit for detection of antigens and/orantibodies); U.S. Pat. No. 6,077,681 (Diagnosis of motor neuropathy bydetection of antibodies); U.S. Pat. No. 6,057,097 (Marker forpathologies comprising an auto-immune reaction and/or for inflammatorydiseases); and U.S. Pat. No. 5,552,285 (Immunoassay methods,compositions and kits for antibodies to oxidized DNA bases).

[0140] By way of example, a microsphere assay (also called flow beadsassays) also can be used to detect Acr protein or another LSA inbiological fluids (such as a culture supernatant from an in vitrolatency model, or biological samples from a subject). This technology,as represented by systems developed by Luminex Corporation and othersystems developed by Becton Dickinson, allows one to process a verysmall amount of sample, typically 20 μl, to detect one or severalanalytes. The principle of this assay is based on the coupling of a“capture antibody” to microspheres containing specific amounts of a reddye and an infrared dye. After incubation of these microspheres with thesample, a secondary detection antibody coupled with biotin andstreptavidin coupled with phycoerthrin (PE), the beads are analyzed witha flow cytometer. One laser detects the beads and a second one detectsthe intensity of the PE bound to those beads. This technology has beenused to detect cytokines in multiplex assays, serotyping ofStreptococcus pneumonia, simultaneous measurement of human chorionicgonadotropin (hCG) and alpha-fetoprotein (AFP), simultaneous detectionof serum IgG to Toxoplasma gondii, Rubella virus, Cytomegalovirus, andHerpes Simplex Virus Types 1 and 2 (see technical notes available fromLuminex Corp., for instance at their Web-site or through their catalog)

[0141] In certain embodiments, a polyclonal rabbit antiserum is used tocapture the Acr protein on the microspheres. In some embodiments, thesecondary detection antibody is a monoclonal antibody to Acr. Secondaryantibodies used in such methods can be coupled to, for instance, biotin.

[0142] VIII. Production of Latency-Specific Immunological BindingPartners

[0143] Once a latency-specific M. tuberculosis protein is identified, itis advantageous to produce that protein, and/or antibodies thatspecifically recognize one or more epitopes on the protein, insufficient amounts to be used in immunological or other assays. Methodsfor production of proteins, and antibodies reactive with identifiedproteins, are well known to those of ordinary skill in the art. Thefollowing methods are provided as representative examples, and shouldnot be viewed as limiting.

[0144] A. Production of Proteins

[0145] Once a latency-specific protein is identified, it is a matter ofwell-known techniques to determine the sequence that encodes theprotein. For instance, the entire coding sequence of the M. tuberculosisgenome is known (Cole et al., Nature 393:537-544, 1998); this can beused to identify the gene that encodes an isolated latency-specificprotein. The encoding sequence can then be used to produce quantities ofprotein in vitro.

[0146] One skilled in the art will understand that there are myriad waysto express a recombinant protein such that it can subsequently bepurified. In general, an expression vector carrying the nucleic acidsequence that encodes the desired protein will be transformed into amicroorganism for expression. Such microorganisms can be prokaryotic(bacteria) or eukaryotic (e.g., yeast). One appropriate species ofbacteria is Escherichia coli (E. coli), which has been used extensivelyas a laboratory experimental expression system. Also, protein can beexpressed using a viral (e.g., vaccinia) based expression system.Protein can also be expressed in animal cell tissue culture, and such asystem will be appropriate where animal-specific protein modificationsare desirable or required in the recombinant protein, or in one portionof a fusion protein.

[0147] Vectors suitable for stable transformation of culturable cellsare also well known. Typically, such vectors include a multiple-cloningsite suitable for inserting a cloned nucleic acid molecule, such that itwill be under the transcriptional control of 5′ and 3′ regulatorysequences. In addition, transformation vectors include one or moreselectable markers; for bacterial transformation this is often anantibiotic resistance gene. Such transformation vectors typically alsocontain a promoter regulatory region (e.g., a regulatory regioncontrolling inducible or constitutive expression), a transcriptioninitiation start site, a ribosome binding site, an RNA processingsignal, and a transcription termination site, each functionally arrangedin relation to the multiple-cloning site. For production of largeamounts of recombinant proteins, an inducible promoter is preferred.This permits selective production of the recombinant protein, and allowsboth higher levels of production than constitutive promoters, andenables the production of recombinant proteins that may be toxic to theexpressing cell if expressed constitutively.

[0148] In addition to these general guidelines, proteinexpression/purification kits are produced commercially. See, forinstance, the QIAexpress™ expression system from QIAGEN (Chatsworth,Calif.) and various expression systems provided by InVitrogen (Carlsbad,Calif.). Depending on the details provided by the manufactures, suchkits can be used for production and purification of latency-specificproteins.

[0149] One skilled in the art will understand that there are myriad waysto purify recombinant polypeptides, and such typical methods of proteinpurification may be used to purify latency-specific proteins. Suchmethods include, for instance, protein chromatographic methods includingion exchange, gel filtration, HPLC, monoclonal antibody affinitychromatography and isolation of insoluble protein inclusion bodies afterover production. In addition, purification affinity-tags, for instance asix-histidine sequence, may be recombinantly fused or linked to theprotein and used to facilitate polypeptide purification. A specificproteolytic site, for instance a thrombin-specific digestion site, canbe engineered into the protein between the tag and the remainder of thefusion to facilitate removal of the tag after purification, if suchremoval is desired.

[0150] Commercially produced protein expression/purification kitsprovide tailored protocols for the purification of proteins made usingeach system. See, for instance, the QIAexpress™ expression system fromQIAGEN (Chatsworth, Calif.) and various expression systems provided byInVitrogen (Carlsbad, Calif.). Where a commercial kit is employed toproduce a functionalized TGF-β fusion protein, the manufacturer'spurification protocol is a preferred protocol for purification of thatprotein. For instance, proteins expressed with an amino-terminalhexa-histidine tag can be purified by binding to nickel-nitrilotriaceticacid Ni-NTA) metal affinity chromatography matrix (The QLIexpressionist,QIAGEN, 1997).

[0151] If the recombinant latency-specific protein is produced in asecreted form, e.g., secreted into the milk of a transgenic animal,purification can be from the secreted fluid. Alternatively, purificationmay be unnecessary if it is appropriate to apply the latency-specificprotein directly to the subject in the secreted fluid (eg., milk), forinstance to induce an immunological response in a subject.

[0152] B. Production of Antibodies

[0153] Monoclonal or polyclonal antibodies may be produced to M.tuberculosis latency-specific proteins, or to specific epitopes withinsuch proteins. Optimally, antibodies raised against a latency-specificprotein would specifically detect that protein. That is, such antibodieswould recognize and bind the Acr protein and would not substantiallyrecognize or bind to other proteins found in a biological sample. Thedetermination that an antibody specifically detects its targetlatency-specific protein is made by any one of a number of standardimmunoassay methods; for instance, the Western blotting technique(Sambrook et al, In Molecular Cloning: A Laboratory Manual, CSHL, NewYork, 1989).

[0154] To determine that a given antibody preparation (such as oneproduced in a mouse) specifically detects the target protein by Westernblotting, total cellular protein is extracted from cells of a latent M.tuberculosis preparation, such as granulomas, and electrophoresed on asodium dodecyl sulfate-polyacrylamide gel. The proteins are thentransferred to a membrane (for example, nitrocellulose) by Westernblotting, and the test antibody preparation is incubated with themembrane. After washing the membrane to remove non-specifically boundantibodies, the presence of specifically bound antibodies is detected bythe use of an anti-mouse antibody conjugated to an enzyme such asalkaline phosphatase. Application of an alkaline phosphatase substrate5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium results inthe production of a dense blue compound by immunolocalized alkalinephosphatase. Antibodies that specifically detect the targetlatency-specific protein will, by this technique, be shown to bind tothe target latency-specific protein band (which will be localized at agiven position on the gel determined by its molecular weight).Non-specific binding of the antibody to other proteins may occur and maybe detectable as a weak signal on the Western blot. The non-specificnature of this binding will be recognized by one skilled in the art bythe weak signal obtained on the Western blot relative to the strongprimary signal arising from the specific antibody-latency-specificprotein binding.

[0155] Substantially pure latency-specific protein suitable for use asan immunogen is isolated from the transfected or transformed cells asdescribed above. Concentration of protein in the final preparation isadjusted, for example, by concentration on an Amicon filter device, tothe level of a few micrograms per milliliter. Monoclonal or polyclonalantibody to the protein can then be prepared as follows:

[0156] i. Monoclonal Antibody Production by Hybridoma Fusion

[0157] Monoclonal antibody to epitopes of a latency-specific proteinidentified and isolated as described (eg., Acr) can be prepared frommurine hybridomas according to the classical method of Kohler andMilstein (Nature 256:495, 1975) or derivative methods thereof. Briefly,a mouse is repetitively inoculated with a few micrograms of the selectedprotein over a period of a few weeks. The mouse is then sacrificed, andthe antibody-producing cells of the spleen are isolated. The spleencells are fused by means of polyethylene glycol with mouse myelomacells, and the excess un-fused cells destroyed by growth of the systemon selective media comprising aminopterin (HAT media). The successfullyfused cells are diluted and aliquots of the dilution placed in wells ofa microtiter plate where growth of the culture is continued.Antibody-producing clones are identified by detection of antibody in thesupernatant fluid of the wells by immunoassay procedures, such as ELISA,as originally described by Engvall (Enzymol. 70:419, 1980), andderivative methods thereof. Selected positive clones can be expanded andtheir monoclonal antibody product harvested for use. Detailed proceduresfor monoclonal antibody production are described in Harlow and Lane(Antibodies, A Laboratory Manual, CSHL, New York, 1988).

[0158] ii. Polyclonal Antibody Production by Immunization

[0159] Polyclonal antiserum containing antibodies to heterogeneousepitopes of a single protein can be prepared by immunizing suitableanimals with the expressed protein, which can be unmodified or modifiedto enhance immunogenicity. Effective polyclonal antibody production isaffected by many factors related both to the antigen and the hostspecies. For example, small molecules tend to be less immunogenic thanothers and may require the use of carriers and adjuvant. Also, hostanimals vary in response to site of inoculations and dose, with eitherinadequate or excessive doses of antigen resulting in low titerantisera. Small doses (ng level) of antigen administered at multipleintradermal sites appear to be most reliable. An effective immunizationprotocol for rabbits can be found in Vaitukaitis et al. (J. Clin.Endocrinol. Metab. 33:988-991, 1971).

[0160] Booster injections can be given at regular intervals, andantiserum harvested when antibody titer thereof, as determinedsemi-quantitatively, for example, by double immunodiffusion in agaragainst known concentrations of the antigen, begins to fall. See, forexample, Ouchterlony et al. (In Handbook of Experimental Immunology,Wier, D. (ed.) chapter 19. Blackwell, 1973). Plateau concentration ofantibody is usually in the range of about 0.1 to 0.2 mg/ml of serum(about 12 μM. Affinity of the antisera for the antigen is determined bypreparing competitive binding curves, as described, for example, byFisher (Manual of Clinical Immunology, Ch. 42, 1980).

[0161] iii. Antibodies Raised Against Synthetic Peptides

[0162] A third approach to raising antibodies against a latency-specificprotein is to use synthetic peptides synthesized on a commerciallyavailable peptide synthesizer based upon the predicted amino acidsequence of the latency-specific protein.

[0163] By way of example only, polyclonal antibodies to specificpeptides within Acr can be generated through well-known techniques byinjecting rabbits with chemically synthesized peptide.

[0164] iv. Antibodies Raised by Injection of Latency-Specific ProteinEncoding Sequence

[0165] Antibodies may be raised against a latency-specific protein bysubcutaneous injection of a DNA vector that expresses thelatency-specific protein into laboratory animals, such as mice. Deliveryof the recombinant vector into the animals may be achieved using ahand-held form of the Biolistic system (Sanford et al., Particulate Sci.Technol. 5:27-37, 1987) as described by Tang et al. (Nature356:152-154,1992). Expression vectors suitable for this purpose mayinclude those that express the Z47 encoding sequence under thetranscriptional control of either the human β-actin promoter or thecytomegalovirus (CMV) promoter.

[0166] Antibody preparations prepared against a latency-specific antigenor epitope of such are useful in quantitative immunoassays thatdetermine concentrations of antigen-bearing substances in biologicalsamples; they are also used semi-quantitatively or qualitatively toidentify the presence of antigen in a biological sample, as describedherein.

[0167] IX. Stimulation of Immunological Responses to Latent Tuberculosis

[0168] With the provision herein of antigens specific to latenttuberculosis infections, methods are now enabled for the stimulation ofimmune responses to such antigens in subjects. In certain embodiments,such immune responses will be protective against formation of latenttuberculosis infection in the subject. Latency-specific proteins (e.g.,Acr) can be used, for instance, as immunogenic agents in the treatment,amelioration, or prevention of latent tuberculosis. Subjects selectedfor this type of treatment are those who are known to have, or aresuspected of having or are at risk of suffering, a latent tuberculosisinfection. An example of such a person is someone who has a positivetuberculin skin test, but has no or limited evidence of active disease(for example clinical symptoms such as fatigue, anorexia, weight loss,fever, nocturnal diaphoresis, cough, hemoptysis, or radiographic orother laboratory evidence recognized as indicative of active disease).

[0169] The provided immunostimulatory proteins or peptides, derived fromlatency-specific proteins (such as Acr) are combined with apharmaceutically acceptable carrier or vehicle for administration as animmunostimulatory composition or a vaccine to human or animal subjects.In some embodiments, more than one protein or peptide fragment may becombined to form a single preparation.

[0170] The immunogenic formulations may be conveniently presented inunit dosage form and prepared using conventional pharmaceuticaltechniques. Such techniques include the step of bringing intoassociation the active ingredient and the pharmaceutical carrier(s) orexcipient(s). In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers. Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example, sealed ampules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of a sterile liquid carrier, for example,water for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets commonly used by one of ordinary skill in the art.

[0171] In certain embodiments, unit dosage formulations are thosecontaining a dose or unit, or an appropriate fraction thereof, of theadministered ingredient. It should be understood that in addition to theingredients particularly mentioned above, formulations encompassedherein may include other agents commonly used by one of ordinary skillin the art.

[0172] The compositions provided herein, including those for use asimmunostimulatory agents or vaccines, may be administered throughdifferent routes, such as oral, including buccal and sublingual, rectal,parenteral, aerosol, nasal, intramuscular, subcutaneous, intradermal,and topical. They may be administered in different forms, including butnot limited to solutions, emulsions and suspensions, microspheres,particles, microparticles, nanoparticles, and liposomes.

[0173] The volume of administration will vary depending on the route ofadministration. By way of example, intramuscular injections may rangefrom about 0.1 ml to 1.0 ml. Those of ordinary skill in the art willknow appropriate volumes for different routes of administration.

[0174] The amount of protein in each vaccine dose is selected as anamount that induces an immunoprotective response without significant,adverse side effects. Such amount will vary depending upon whichspecific immunogen is employed and how it is presented. Initialinjections may range from about 1 μg to 1 mg, with some embodimentshaving a range of about 10 μg to 800 μg, and still other embodiments arange of from approximately 25 μg to 500 μg. Following an initialvaccination, subjects may receive one or several booster immunizations,adequately spaced. Booster injections may range from 1 μg to 1 mg, withother embodiments having a range of approximately 10 μg to 750 μg, andstill others a range of about 50 μg to 500 μg. Periodic boosters atintervals of 1-5 years, for instance three years, may be desirable tomaintain the desired levels of protective immunity.

[0175] As described in WO 95/01441, the course of the immunization maybe followed by in vitro proliferation assays of PBL (peripheral bloodlymphocytes) co-cultured with ESAT6 or ST-CF, and especially bymeasuring the levels of IFN-released from the primed lymphocytes. Theassays are well known and are widely described in the literature,including in U.S. Pat. Nos. 3,791,932; 4,174,384 and 3,949,064.

[0176] A recent development in the field of immune stimulatory compounds(e.g., vaccines) is the direct injection of nucleic acid moleculesencoding peptide antigens (broadly described in Janeway & Travers,Immunobiology: The Immune System In Health and Disease, page 13.25,Garland Publishing, Inc., New York 1997; and McDonnell & Askari, N. Eng.J. Med. 334:42-45, 1996). Plasmids that include nucleic acid moleculesdescribed herein, or that include a nucleic acid sequence encoding animmunogenic peptide or peptide fragment of a latency specificpolypeptide (such as Acr) or derived from a latency specific polypeptide(for instance, as a fusion protein, may be utilized in such DNAvaccination methods.

[0177] Thus, the terms “immunostimulatory preparation” and “vaccine” asused herein also include nucleic acid vaccines in which a nucleic acidmolecule encoding a latency-specific polypeptide (such as Acr), or afragment thereof is administered to a subject in a pharmaceuticalcomposition. For genetic immunization, suitable delivery methods knownto those skilled in the art include direct injection of plasmid DNA intomuscles (Wolff et al., Hum. Mol. Genet. 1:363, 1992), delivery of DNAcomplexed with specific protein carriers (Wu et al., J. Biol. Chem.264:16985, 1989), co-precipitation of DNA with calcium phosphate(Benvenisty and Reshef, Proc. Natl. Acad. Sci. 83:9551, 1986),encapsulation of DNA in liposomes (Kaneda et al., Science 243:375,1989), particle bombardment (Tang et al., Nature 356:152, 1992) and(Eisenbraun et al., DNA Cell Biol. 12:791, 1993), and in vivo infectionusing cloned retroviral vectors (Seeger et al., Proc. Natl. Acad. Sci.81:5849, 1984).

[0178] Similarly, nucleic acid vaccine preparations can be administeredvia viral carrier.

[0179] It is also contemplated that the provided immunostimulatorymolecules and preparations can be administered to a subject indirectly,by first stimulating a cell in vitro, which stimulated cell isthereafter administered to the subject to elicit an immune response.

[0180] X. Immunological and Pharmaceutical Compositions

[0181] Immunological compositions, including immunological elicitorcompositions and vaccines, and other pharmaceutical compositionscontaining latency-specific polypeptides or antigenic fragments thereofare useful for reducing, ameliorating, treating, or possibly preventingmycobacterial infection, particularly latent M. tuberculosis infection.One or more of the polypeptides are formulated and packaged, alone or incombination with adjuvants or other antigens, using methods andmaterials known to those skilled in the vaccine art. An immunologicalresponse of a subject to such an immunological composition may be usedtherapeutically or prophylactically, and in certain embodiments providesantibody immunity and/or cellular immunity such as that produced by Tlymphocytes such as cytotoxic T lymphocytes or CD4⁺ T lymphocytes.

[0182] To enhance immunogenicity, one or more immunogenic polypeptidesor fragments (e.g., haptens) may be conjugated to a carrier molecule.Immunogenic carrier molecules include proteins, polypeptides or peptidessuch as albumin, hemocyanin, thyroglobulin and derivatives thereof,particularly bovine serum albumin (BSA) and keyhole limpet hemocyanin(KLH), polysaccharides, carbohydrates, polymers, and solid phases. Otherprotein-derived or non-protein-derived substances are known to those ofordinary skill in the art. An immunogenic carrier typically has amolecular weight of at least 1,000 Daltons, and in some embodimentsgreater than 10,000 Daltons. Carrier molecules often contain a reactivegroup to facilitate covalent conjugation to the hapten. The carboxylicacid group or amine group of amino acids or the sugar groups ofglycoproteins are often used in this manner. Carriers lacking suchgroups can often be reacted with an appropriate chemical to producethem. Alternatively, a multiple antigenic polypeptide comprisingmultiple copies of the protein or polypeptide, or an antigenically orimmunologically equivalent polypeptide may be sufficiently antigenic toimprove immunogenicity without the use of a carrier.

[0183] The latency-specific polypeptides may be administered with anadjuvant in an amount effective to enhance the immunogenic responseagainst the conjugate. At this time, the only adjuvant widely used inhumans has been alum (aluminum phosphate or aluminum hydroxide). Saponinand its purified component Quil A, Freund's complete adjuvant and otheradjuvants used in research and veterinary applications have toxicitieswhich limit their potential use in human vaccines. However, chemicallydefined preparations such as muramyl dipeptide, monophosphoryl lipid A,phospholipid conjugates such as those described by Goodman-Snitkoff etal. (J. Immunol. 147:410-415, 1991), encapsulation of the conjugatewithin a proteoliposome as described by Miller et al. (J. Exp. Med176:1739-1744, 1992), and encapsulation of the protein in lipid vesiclesmay also be useful.

[0184] The compositions provided herein, including those formulated toserve as vaccines, may be stored at temperatures of from about −100° C.to 4° C. They may also be stored in a lyophilized state at differenttemperatures, including higher temperatures such as room temperature.The preparation may be sterilized through conventional means known toone of ordinary skill in the art. Such means include, but are notlimited to filtration, radiation and heat. The preparations also may becombined with bacteriostatic agents, such as thimerosal, to inhibitbacterial growth.

[0185] A variety of adjuvants known to one of ordinary skill in the artmay be administered in conjunction with the protein(s) in the providedvaccine composition. Such adjuvants include but are not limited to thefollowing: polymers, co-polymers such aspolyoxyethylene-polyoxypropylene copolymers, including blockco-polymers; polymer P1005; Freund's complete adjuvant (for animals);Freund's incomplete adjuvant; sorbitan monooleate; squalene; CRL-8300adjuvant; alum; QS 21, muramyl dipeptide; CpG oligonucleotide motifs andcombinations of CpG oligonucleotide motifs; trehalose; bacterialextracts, including mycobacterial extracts; detoxified endotoxins;membrane lipids; or combinations thereof.

[0186] In a particular embodiment, a vaccine is packaged in a singledosage for immunization by parenteral (i.e., intramuscular, intradermalor subcutaneous) administration or nasopharyngeal (i.e., intranasal)administration. In certain embodiments, the vaccine is injectedintramuscularly into the deltoid muscle. The vaccine may be combinedwith a pharmaceutically acceptable carrier to facilitate administration.The carrier is, for instance, water, or a buffered saline, with orwithout a preservative. The vaccine may be lyophilized for resuspensionat the time of administration or in solution.

[0187] The carrier to which the polypeptide may be conjugated may alsobe a polymeric delayed release system. Synthetic polymers areparticularly useful in the formulation of a vaccine to effect thecontrolled release of antigens.

[0188] Microencapsulation of the polypeptide will also give a controlledrelease. A number of factors contribute to the selection of a particularpolymer for microencapsulation. The reproducibility of polymer synthesisand the microencapsulation process, the cost of the microencapsulationmaterials and process, the toxicological profile, the requirements forvariable release kinetics and the physicochemical compatibility of thepolymer and the antigens are all factors that must be considered.Examples of useful polymers are polycarbonates, polyesters,polyurethanes, polyorthoesters polyamides,poly-(d,l-lactide-co-glycolide) (PLGA) and other biodegradable polymers.

[0189] Doses for human administration of a pharmaceutical composition ora vaccine may be from about 0.01 mg/kg to 10 mg/kg, for instanceapproximately 1 mg/kg. Based on this range, equivalent dosages forheavier (or lighter) body weights can be determined. The dose may beadjusted to suit the individual to whom the composition is administered,and may vary with age, weight, and metabolism of the individual, as wellas the health of the subject. Such determinations are left to theattending physician or another familiar with the subject and/or thespecific situation. The vaccine may additionally contain stabilizers orphysiologically acceptable preservatives, such as thimerosal(ethyl(2-mercaptobenzoate-S)mercury sodium salt) (Sigma Chemical Co.,St. Louis, Mo.).

[0190] XI. Kits

[0191] Kits are provided which contain the necessary reagents forgrowing in vitro granulomas or for determining the presence (or absence)of a latency-specific antigen and/or antibody in a biological sample,using an immunological binding reaction. Instructions provided in thediagnostic kits can include calibration curves, diagrams, illustrations,or charts or the like to compare with the determined (e.g.,experimentally measured) values or other results.

[0192] A. Kits for Growing In Vitro Granulomas

[0193] Kits for growing in vitro granulomas include, for instance, cellculture media (e.g. RPMI plus 10% human serum, HS) and optionally mayinclude a low attachment container (e.g., a tissue culture dish treatedto inhibit all attachment), a filter, and/or a fixative. Specificexamples of such kits also include an amount of one or more cytokine,for instance IL-2, IFN-γ, and/or TNF-α. Reagents supplied in the kitsmay be contained in separate containers.

[0194] The kits may also include means for granuloma analysis, forinstance ELISA reagents, reagents for RT-PCR, and/or RPA reagents, whichmay also be provided in some kits in one or more separate containers.Cell culture, ELISA, RT-PCR, and RPA techniques are well known to thoseof ordinary skill in the art.

[0195] Reaction vessels and auxiliary reagents such as buffers, enzymes,etc. may also be included in the kits.

[0196] Additional components in some kits include instructions forcarrying out the cell culture and/or subsequent analysis. Whereprovided, instructions may allow the tester to grow in vitro granulomasand use them to identify latency-specific antigens and screen drugs andimmunostimulatory compounds, such as vaccines.

[0197] B. Kits for Detection of Latency-Specific Antigen

[0198] Kits for the detection of latency-specific M. tuberculosisprotein expression include for instance at least one target proteinspecific binding agent (e.g., a polyclonal or monoclonal antibody orantibody fragment) and may include at least one control. Thelatency-specific protein specific binding agent and control may becontained in separate containers. The kits may also include means fordetecting target protein agent complexes, for instance the agent may bedetectably labeled. If the detectable agent is not labeled, it may bedetected by second antibodies or protein A, for example, which may alsobe provided in some kits in one or more separate containers. Suchtechniques are well known.

[0199] Additional components in some kits include instructions forcarrying out the assay. Instructions will allow the tester to determinewhether latency-specific protein expression levels are altered, forinstance in comparison to a control sample. Reaction vessels andauxiliary reagents such as chromogens, buffers, enzymes, etc. may alsobe included in the kits.

[0200] By way of example only, an effective and convenient immunoassaykit such as an enzyme-linked immunosorbent assay can be constructed totest anti-Acr antibody in human serum. Expression vectors can beconstructed using the Acr cDNA to produce the recombinant Acr protein ineither bacteria or baculovirus (as described above). By affinitypurification, unlimited amounts of pure recombinant latency-specificprotein (such as Acr) can be produced.

[0201] C. Kits for Detection of Antibody to Latency-Specific Antigens

[0202] Other examples of assay kits provide a recombinantlatency-specific protein as an antigen and enzyme-conjugated goatanti-human IgG as a second antibody. Examples of such kits also caninclude one or more enzymatic substrates. Such kits can be used to testif a biological sample from a subject contains antibodies against alatency-specific protein.

[0203] The disclosure is further illustrated by the followingnon-limiting Examples.

EXAMPLES Example 1 Preparation and Characterization of an In VitroGranuloma Model

[0204] The principal defense of the human host against a mycobacterialinfection is the formation of granulomas, which are compact organizedcollections of activated macrophages, including epithelioid andmultinucleated giant cells, surrounded by T lymphocytes, and later byfibroblasts and collagen that aggregate around the macrophage-core. Thegranuloma may prevent active (non-latent) disease by sequestering theinvading organisms. If the granuloma is maintained, these bacteria mayremain latent for many years.

[0205] To study this process of granuloma formation and the granuloma'ssubsequent breakdown when host defenses are compromised, an in vitromodel was developed. This example provides a description of one methodfor producing the in vitro granuloma that can be used as a model system,as well as several methods used to characterize the model. In overview,human peripheral blood mononuclear cells, autologous macrophages andmycobacteria were combined in low attachment tissue culture dishes. Theresulting aggregates were characterized using microscopy andimmunohistochemical staining. Cytokine production was assessed by ELISAand bacterial mRNA detected by RT-PCR.

[0206] Peripheral blood mononuclear cells (1×10⁶), autologousmacrophages (1×10⁶) and mycobacteria (1×10¹) were combined in lowattachment tissue culture dishes (COSTAR™ Ultra Low Attachment Clusters,Costar Corp., Cambridge, Mass.) and incubated at 37° C. in 5% CO₂. Humanperipheral blood mononuclear cells (PBMCs, 1×10⁶ cells in RPMI plus 10%HS) were added after 24 hours, and the mixture incubated for 5-7 days.Aggregate formation was observed. Human lung fibroblasts (from the cellline 33Lu) were added when the aggregates were approximately 1 mm indiameter.

[0207] The aggregates were characterized using microscopy andimmunohistochemical staining with standard methodology. Small, roundedaggregate structures were formed in the cultures, which developed moredefined edges with the addition of human lung fibroblasts. Microscopicexamination of these aggregates using immunostaining found CD68⁺epithelioid macrophages and sparse, small round CD3⁺ lymphocytes than incomplex, resembled small granulomas seen in clinicopathologic specimens.Acid-fast staining bacteria were observed within and between the cellscomposing the granulomas (FIG. 1).

[0208] In addition to morphology, cytokine production was assessed byELISA. In particular, cytokines that are known to be upregulated duringearly stage M. tuberculosis infection were analyzed.

[0209] By 24 hours following infection, the aggregates were found togenerate levels of TNF, IL-8, and IL-6 that-were elevated well abovelevels found in uninfected control cells. After 48 hours levels of IFNwere likewise increased above controls. This elevated cytokineproduction continued over the nine day duration of the experiment.Results also indicate increased levels of IL-2 and IL-12, peaking at 48hours, but remaining above control levels throughout the course of theexperiment. All of these cytokines are detected at significantly higherlevels in tuberculosis patients when compared to healthy controls.

Example 2 Detection of Differential Transcription of Acr mRNA in aGranuloma Model

[0210] Using bacteria cultured in an anoxic chamber, M. tuberculosisgenes were identified that were differentially expressed after much ofthe available oxygen had been utilized. The genes that weredifferentially expressed were acr, sigF, oxyR and aphC. Of these fourgenes, acr encodes a protein (α-crystallin) that is secreted by theMycobacterium.

[0211] In order to confirm that these genes are expressed in the invitro granuloma model, RT-PCR and RNA protection assays were performed.These assays showed that mRNA from mycobacterial genes acr, aphC, andsigF were transcribed. These transcripts were not found in uninfected invitro granuloma controls.

[0212] Typical results from representative experiments are shown inFIGS. 2 and 3, which are ribonuclease protection assay (RPA) blots. InFIG. 2, acr mRNA was observed at all four time points while RpoB mRNAwas only observed in aerobically grown cultures. In FIG. 3, acr and RpoBmRNA were observed both at 7- or 12-day incubations in the in vitrogranuloma model, which is believed to indicate that aerobic bacilli werepresent in the granuloma

Example 3 Other In Vitro Latency Models

[0213] The following example provides other in vitro latency models,which can be used, for example, to confirm results obtained with the invitro granuloma model (e.g., to screen drugs and immunostimulatorycompounds and to identify latency-specific antigens).

[0214] Guinea Pig Aerosol Infection Model.

[0215] When infected by aerosol inoculation using a small number of M.tuberculosis bacilli, it was observed that the bacteria cause formationof granulomas associated with the epithelial pneumocytes in the deepalveoli of the lung. Though these granulomas apparently are not able tocompletely contain the infection, and the bacteria eventually overwhelmthis animal, there are a number of similarities with human granulomas.For example, these granulomas center on necrotic areas and containpredominantly macrophages, including epithelioid and multinucleatedgiant cells and T-lymphocytes. Differential transcription of thebacterial Acr gene has been detected in these granulomas.

[0216] In Vitro Anoxic Chamber Models.

[0217] Wayne and Hayes developed an in vitro persistence model thatsubject M. tuberculosis bacilli to gradual oxygen deprivation byincubation in sealed containers with controlled agitation (Wayne andHayes, Infect. Immun. 64:2062-2069, 1996). Growth under these conditionscan be operationally divided into two non-replicating persistent (NRP)states: a microacrophilic state associated with induction of glycinedehydrogenase activity (NRP1), and a subsequent lower oxygen state(NRP2), in which glycine dehydrogenase activity declines and alterationsin drug susceptibility are manifest. Specifically, cells becomeresistant to ciprofloxicin and sensitive to metronidazole, possible dueto changes in DNA superhelicity and cell permeability, respectively(Wayne and Hayes, Infect. Immun. 64:2062-2069, 1996). These observationscorrelate with the intractability of clinical TB to single antibiotictherapy (Dickinson and Mitchison, Am. Rev. Respir. Dis. 123:367-371,1981).

[0218] A modified version of this sealed vessel has been developed (FIG.5), which allows monitoring of several environmental growth conditions(including optical density culture population, oxygen concentration, pH,and assaying of enzymes induced only under low oxygen tension), as wellas providing containment of the pathogen and easy nucleic acid harvestby direct centrifugation of the vessel. Using this system, it has beenshown that M. tuberculosis bacilli cease to replicate but remainmetabolically active for several months under conditions of low oxygen(FIG. 12).

Example 4 Construction of Acr-FLAG Fusion Proteins

[0219] Oligonucleotide primers for polymerase chain reaction (PCR) weredesigned and generated. These primers were designed to allow theamplification of the hspX gene from M. tuberculosis (encoding Acr) withthe addition of the FLAG (Sigma) epitope tag fused to amino- orcarboxy-terminus of Acr depending on which primer pair was used. Primerdesign also included the introduction of restriction endonucleaserecognition sites to facilitate subsequent recombinant DNA methodologiesinvolved in cloning and expression of these amplified sequences.Sequences of the primers to generate the N-terminal FLAG-Acr fusion wereSEQ ID NO: 1 and SEQ ID NO: 2. Sequences of the primers to generate theC-terminal Acr-FLAG fusion were SEQ ID NO: 3 and SEQ ID NO: 4.

[0220] PCR reactions were performed using one microgram MTB H37Rvgenomic DNA as template and 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mMMgCl₂, and 2.5 units AmpliTaq (Gibco BRL) thermostable DNA polymerase.PCR was performed with the following cycle parameters: 94° C., 5 minutes(1×), 94° C., 1 minute; 55° C., 30 seconds; 72° C., 2.5 minutes (2×),94° C., 1 minute; 60° C., 30 seconds; 72° C., 2.5 minutes (30×), 72° C.,7 minutes (1×). Following PCR amplification the amplified DNA fragmentswere purified and digested by restriction endonuclease, and ligated intolikewise digested plasmid vector pMV261.1. Following ligation, thereactions were transformed into E. coli and positive colonies selectedby antibiotic resistance. Selected colonies were screened for thepresence of the recombinant insert by PCR, and sequence verification wasachieved by DNA sequencing of each positive clone. Positive N-terminaland C-terminal Act-FLAG fusion plasmids were subsequently transformedinto both M. smegmatis and M. tuberculosis and transformed coloniesselected by antibiotic resistance.

[0221] Expression of the fusion proteins was confirmed by Westernblotting; representative blots are shown in FIG. 6 (N-terminal fusion)and FIG. 7 (C-terminal fusion).

Example 5 Immunoprecipitation of Acr

[0222] Acr-FLAG fusion proteins were immunoprecipitated with a FLAGImmunoprecipitation Kit acquired from Sigma according to themanufacturer's instructions. Native Acr immunoprecipitations wereperformed on mycobacterial lysates using sepharose CL4B beads(Pharmacia) to which polyclonal rabbit antibodies generated against Acrhad been conjugated according to the manufacturer's instructions.Briefly, a pre-clearing step was performed to remove any proteins fromthe lysate that might non-specifically bind to the sepharose beads. Thiswas accomplished by adding 50 μl of unconjugated sepharose to 975 μl ofTSA (0.01M Tris-HCl, pH 8; 0.14M NaCl; 0.025% NaN₃) and 16 μl of MTBlysate (2.3 mg/mL) in a 1.5 mL microfuge tube. The solution wasincubated for one hr at 4° C. with constant rocking. The solution wasthen centrifuged for 5 sec at 14,000 rpm to pellet the sepharose and thesupernatant removed and transferred to a new microfuge tube. 25 μl ofanti-Acr antibody conjugated sepharose beads were added to thesupernatant and incubated for one hour at 4° C. with constant rocking.The solution was then centrifuged as previously described and thesupernatant was removed and discarded. The sepharose pellet was thenwashed 4 times with 1 mL each of 1) 0.1% Triton X-100 in TSA, 2) 0.1%Triton X-100 in TSA, 3) TSA, 4) 0.05M Tris-HCl, pH 6.8. Addition of eachsolution was followed by resuspension of the sepharose pellet, a5-second centrifugation to repellet the sepharose, and removal of thewash supernatant. Finally, 40 μl of 2× gel loading buffer (Novex) wasadded to the sepharose pellet, and the suspension incubated for 1.5hours at 56° C. A final centrifugation was performed to re-pellet thesepharose and the supernatant (containing the immunoprecipitated Acrprotein) was removed and saved for analysis by Western blot.

Example 6 Western Blot

[0223] Immunoprecipitated Acr samples or mycobacterial lysates wereresolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS PAGE) with pre-cast 412% bis-Tris polyacrylamide gradient gels(Novex) for one hour at 200 volts constant. Following electrophoresis,the resolved proteins were transferred to a pre-cut 0.2-micronpolyvinylidene difluoride membrane (VDF, Novex) according to themanufacturer's instructions by electrocapillary transfer at 150milliamps constant for one hour. Dried membranes were prepared for Acrdetection by immersion in methanol and unconjugated PVDF surfaces wereblocked by incubation of the membrane in TBS Tween 20 containing 3%gelatin (BioRad) for one hour with constant rocking. The membrane wasthen washed 3 times for 10 minutes each with TBS+Tween 20 with constantrocking.

[0224] Detection of Acr was accomplished by incubation of the membranewith a 1 to 5,000 dilution of polyclonal rabbit antibodies generatedagainst Acr, or a 1:20,000 anti-FLAG M2 monoclonal antibody, (Sigma), ora 1 to 250 dilution of rabbit anti-phosphotyrosine antibody (Zymed) inTBS+Tween 20 containing 1% gelatin for one hour with constant rocking.Subsequently, the membrane was washed as described above. Aone-to-10,000 dilution of biotinylated goat-anti-rabbit IgG in TBS+Tween20 with 1% gelatin was then applied to the blot, and incubated for onehour with constant rocking. Subsequently, the blot was washed asdescribed above. A final incubation of the blot with a 1 to 10,000dilution of a streptavidin-alkaline phosphatase (AP) conjugate inTBS+Tween 20 was incubated with the blot for 30 minutes with constantrocking. Detection of the presence of AP was accomplished by theincubation of the blot in a solution of NBT+BCIP (Novex). The incubationwas halted when the desired level of color development was achieved, andstopped by extensive washing of the blot in water. Blots were dried forarchiving.

[0225]FIG. 8 shows five strips cut from a Western blot prepared asdescribed of whole cell mycobacteria cell lysates; the arrows indicatethe location of recombinant Acr protein. The strips were developed withthe indicated primary antibodies. In addition to the control antibody,this blot demonstrates the presence of tyrosine phosphorylation in thefinal strip.

[0226]FIG. 9 shows two Western blots prepared as described from culturesupernatants of M. tuberculosis bacilli grown under the conditionsindicated in the Brief Description of the Figures. FIG. 9A was probedusing rabbit anti-Acr antibody; FIG. 9B is a control blot Acr protein isdetected in 7 day and 12 month anoxic cultures and in vitro granuloma.Lower molecular weight variants are observed in the 12 month and invitro granuloma supernatants. For comparison, FIG. 11 shows s aCoomassie stained SDS-PAGE gel of culture supernatants from M.tuberculosis bacilli cultured under the indicated conditions.

Example 7 2 Dimensional Gel Electrophoresis

[0227] Lysates of M. smegmatis and M. tuberculosis carrying the Acr-FLAGfusion plasmids were prepared. Bacterial suspension cultures werepelleted by centrifugation (5,000 rpm, 10 minutes) and the bacterialpellet washed with phosphate buffered saline (PBS). Pellets were thenresuspended in 1 mL of 9M urea and the suspension transferred to 2 mLmicrofuge tubes containing approximately 200 μl of 0.1 mm diameter glassbeads (Biospec products). The cells were then lysed by rapid shaking(three times, one minute each) in a Minibeadbeater Biospec products).Following lysis, the cellular debris and glass beads were pelleted bycentrifugation and the supernatant removed. Protein quantification wasperformed using the colorimetric BioRad protein assay (BioRad).

[0228] For resolution of the protein sample in the first dimension, 150micrograms of protein was added to 92.5 μl of solubilization buffer (9MUrea, 140 mM DTT, 4% Triton X-100) and brought to a final volume of 185μL with 9M urea. The suspension was incubated for one hour with constantrocking at room temperature. Following this incubation, 1 μl Biolyteselectrolyte solution (BioRad, 0.2% final), and several crystals ofBromophenol Blue (Fisher) were added. Hydration of the IPG strips andapplication of the protein solution to the strips was carried outovernight in a IPG strip hydration apparatus (Pharmacia). Followinghydration the strips were subjected to electrophoresis in a LKBMultiPhor II apparatus (Pharmacia) using a Pharmacia EPS 3500 XLelectrophoresis apparatus with the following parameters: 350V, 30minutes, 350V-3500V (1.5 hour gradient), 3500V, 3.5 hours, to give afinal running time of 15,200 Volt-hours.

[0229] For running the second dimension, the resolved IPG strips wereequilibrated in equilibration buffer (6M Urea, 0375M Tris, pH 8.8, 2%SDS, and 20% Glycerol) containing 2% w/v DTT (10 minutes) and 2.5%iodoacetamide (10 minutes). The equilibrated strips were resolved onCriterion (BioRad) 8-16% polyacrylamide gels according to themanufacturer's instructions for one hour at 200 volts constant.

[0230]FIG. 10 is a representative two-dimensional gel electrophoresisanalysis of a sample taken from M. tuberculosis grown under anoxicconditions. Acr protein is indicated by the circle.

Example 8 Detection of Acr

[0231] Using polyclonal antibodies produced against the cloned M.tuberculosis Acr protein, secreted bacterial Acr was-detected in culturesupernatants from anoxic chamber and the in vitro granuloma model (FIG.9). Polyclonal antiserum to Acr protein was also used to detectmycobacteria in aerosol infected guinea pig lung granuloma tissue (FIG.1C).

[0232] Acr protein was detected in supernatants and cell lysates fromanoxic but not aerobic grown M. tuberculosis (FIG. 9). Acr wasimmunoprecipitated from M. tuberculosis lysates. Changes in molecularweight of Acr were detected in anoxic or infected in vitro granulomasbut not aerobic grown cultures (FIG. 9). M. tuberculosis acr mRNA wasdetected in anoxic but not aerobic cultures and acr and eukaryotic actinmRNA in infected but not uninfected control guinea pig lungs (FIG. 4).

[0233] This disclosure provides an in vitro granuloma model and methodsof use, as well as immunological methods for the detection of latenttuberculosis in a subject. The in vitro granuloma model can be used, forexample, to identify latency-specific antibodies and to screen drugs andimmunostimulatory compounds. The immunological methods can include, forexample, using a latency-specific M. tuberculosis antigen to detect acorresponding antibody from the subject, or using an antibody to detectthe latency-specific antigen. The disclosure further provides methodsfor identifying latency-specific antigens (and their correspondingantibodies) for use in such methods, and specific latency-specificantigens such as α-crystallin. It will be apparent that the precisedetails of the methods described may be varied or modified withoutdeparting from the spirit of the described disclosure. We claim all suchmodifications and variations that fall within the scope and spirit ofthe claims below.

1 4 1 54 DNA Artificial sequence Synthetic oligonucleotide 1 cgcggatccagattataaag atgatgatga taaaatggcc accacccttc ccgt 54 2 33 DNA Artificialsequence Synthetic oligonucleotide 2 cgcggatcct cagttggtgg accggatctgaat 33 3 29 DNA Artificial sequence Synthetic oligonucleotide 3cgcggatcca atggccacca cccttcccg 29 4 57 DNA Artificial sequenceSynthetic oligonucleotide 4 cgcggatcct catttatcat catcatcttt ataatcgttggtggaccgca tctgaat 57

We claim:
 1. An immunological assay method for detection of latenttuberculosis in a subject, comprising contacting a biological samplethat may contain a first latency-specific binding partner (LSBP) fromthe subject with a corresponding LSBP, and detecting binding between thefirst LSBP and the corresponding LSBP, wherein such binding isindicative of latent tuberculosis in the subject.
 2. The method of claim1, wherein the first LSBP is an antibody, and the corresponding LSBP isa latency-specific M. tuberculosis antigen.
 3. The method of claim 2,wherein the antigen is α-crystallin (Acr) or an immunogenic fragmentthereof.
 4. The method of claim 1, wherein the first LSBP is alatency-specific M. tuberculosis antigen, and the corresponding LSBP isan antibody.
 5. The method of claim 4, wherein the antigen is Acr or animmunogenic fragment thereof.
 6. A kit for the detection of latenttuberculosis in a subject, comprising at least one LSBP and instructionsfor carrying out the method of claim
 1. 7. A method of eliciting animmune response in a subject, comprising introducing into the subject animmune stimulatory amount of a M. tuberculosis latency-specific antigenor immunogenic fragment thereof, or a nucleic acid molecule encodingsuch an antigen or immunogenic fragment.
 8. The method of claim 7, whichis a method of inhibiting a latent tuberculosis infection in thesubject.
 9. The method of claim 7, which is a method of treating alatent tuberculosis infection in the subject.
 10. The method of claim 7,wherein the antigen is Acr.
 11. The method of claim 7, wherein theelicited immune response results in decreased susceptibility of thesubject to latent infection by M. tuberculosis.
 12. A kit for elicitingan immune response in a subject, comprising an immune stimulatory amountof a M. tuberculosis latency-specific antigen or immunogenic fragmentthereof, or a nucleic acid molecule encoding such an antigen orimmunogenic fragment, and instructions for carrying out the method ofclaim
 7. 13. The kit of claim 12, further comprising instructions foradministering a component of the kit to a patient with a possible latenttuberculosis infection.
 14. An in vitro granuloma comprising peripheralblood mononuclear cells, autologous macrophages, and mycobacteria. 15.The in vitro granuloma of claim 14, wherein the peripheral bloodmononuclear cells are human peripheral blood mononuclear cells selectedfrom the group consisting of monocytes, B lymphocytes, T lymphocytes,and combinations thereof.
 16. The in vitro granuloma of claim 14,wherein the mycobacteria are M. tuberculosis.
 17. The in vitro granulomaof claim 14, further comprising fibroblasts.
 18. A method for producingan in vitro granuloma comprising combining peripheral blood mononuclearcells, autologous macrophages, and mycobacteria in a low attachmentcontainer and incubating the combination for a sufficient amount of timeto form the in vitro granuloma.
 19. The method of claim 18, whereinfibroblasts are added to the combination.
 20. The method of claim 18,further comprising adding exogenous cytokine to the container insufficient amount to enhance production of the in vitro granuloma. 21.The method of claim 20, wherein the exogenous cytokine is IL-2, IFN-γ,TNF-α, or a combination of two or more thereof.
 22. A method ofscreening a tuberculosis drug candidate for anti-tuberculosistherapeutic activity comprising combining the drug with an in vitrogranuloma comprising peripheral blood mononuclear cells, autologousmacrophages, and mycobacteria, and determining whether the drug inhibitsmycobacterial viability.
 23. The method of claim 18 or claim 22, whereinthe peripheral blood mononuclear cells are human peripheral bloodmononuclear cells selected from the group consisting of monocytes, Blymphocytes, T lymphocytes, and combinations thereof.
 24. A method ofscreening a tuberculosis drug candidate for anti-tuberculosistherapeutic activity comprising combining the drug with an in vitrogranuloma comprising peripheral blood mononuclear cells, autologousmacrophages, and inactivated mycobacteria, and determining whether thedrug inhibits reactivation of mycobacteria contained in the granuloma.25. The method of claim 18, claim 22, or claim 24, wherein themycobacteria are M. tuberculosis.
 26. A method of screening atuberculosis vaccine candidate comprising determining whether a mutantmycobacteria has a reduced ability, when compared against a wild typemycobacteria, to induce latency, survive, reactivate or induce granulomanecrosis in an in vitro granuloma comprising peripheral bloodmononuclear cells, autologous macrophages, and the mutant mycobacteria.27. The method of claim 22, claim 24, or claim 26, wherein the in vitrogranuloma further comprises fibroblasts.
 28. The method of claim 26,wherein the mutant mycobacteria comprises a mycobacteria strain having amutation in a latency gene.
 29. The method of claim 26, wherein themutant mycobacteria is a Mycobacterium tuberculosis strain having amutation in a gene selected from the group consisting of acr, a sigmafactor gene, oxyR and aphC.
 30. The method of claim 29, wherein thesigma factor gene is selected from the group consisting of sigF, sigC,and sigH.
 31. A kit for producing an in vitro granuloma, comprising aculture medium and instructions for carrying out the method of any oneof claims 18, 22, 24, or
 26. 32. The kit of claim 31, further comprisinga low attachment container.
 33. The kit of claim 31, further comprisingan amount of a cytokine.